Vaccine compositions comprising C—C motif chemokine 22 (CCL22) or fragments thereof

ABSTRACT

The present disclosure relates to CCL22 as a novel T cell target in cancer immunosuppression.

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created Sep. 20, 2018, isnamed “Corrected Sequence Listing N414655US ST25.txt” and is 4707 bytesin size.

FIELD OF INVENTION

The present invention relates to the field of prophylaxis and therapy ofcancer. In particular there is provided a protein C-C motif chemokine 22(CCL22) or peptide fragments here of that are capable of elicitinganti-cancer immune responses. Specifically, the invention relates to theuse of CCL22 or peptides derived thereof or CCL22 specific T-cells fortreatment of cancer. The invention thus relates to an anti-cancervaccine which optionally may be used in combination with otherimmunotherapies and to CCL22 specific T-cells adoptively transferred orinduced in vivo by vaccination as a treatment of cancer. It is an aspectof the invention that the medicaments herein provided may be used incombination with cancer chemotherapy treatment. A further aspect relatesto the prophylaxis and therapy of infections by the same means asdescribed above.

The use of CCL22 and immunogenic peptide fragments hereof in cancer andinfection treatment, diagnosis and prognosis is also provided.

BACKGROUND OF INVENTION

The immune system is a complex arrangement of cells and molecules thatpreserve the integrity of the organism by the elimination of allelements that are judged to be dangerous. Several regulatory mechanismsfunction to terminate immune responses to antigens, returning the immunesystem to a basal state after the antigen has been cleared, and tomaintain the unresponsiveness, or tolerance, to self-antigens.Unfortunately, some of the mechanisms preventing autoimmunity arehijacked by cancers to attain immune escape. Indeed, in ‘The Hallmarksof cancer’ by Hanahan and Weinberg, evasion of immune destruction is nowlisted as an emerging hallmark (Nomi et al., 2007). This evasion ofimmune destruction is based on several mechanisms. Solid tumors arecomposed of the cancerous cells themselves as well as the stroma thatnot only provides a supportive framework but also allows immune evasion.For this purpose, tumors attract and/or convert immune competent cellsthat procreate and sustain an immune permissive microenvironment. Theseimmune competent cells, which are normally involved in the elaboratenetwork of central and peripheral tolerance mechanisms maintainingimmune homeostasis, include factor forkhead box P3 (Foxp3)-positiveregulatory T cells (Tregs), NKT cells, dendritic cell subtypes, myeloidderived suppressor cells (MDSC), M2 (aka tumor-associated) macrophages,and granulocytes. The mechanism by which CCL22 is thought to act incancer is illustrated in FIG. 1.

This immune evasion is harmful in the context of cancer immunotherapy.Thus, the targeting of one or more immunosuppressive pathways may behighly useful in combination with anti-cancer immunotherapy in whichimmune-suppressive mechanisms antagonize the required effects. Severaldifferent potential therapeutic strategies with which to target immunesuppression in cancer are under investigation, especially the blockingof inhibitory pathways using monoclonal antibodies.

SUMMARY OF INVENTION

The invention is as defined in the claims.

The expression of CCL22 and the potential CCL22-induced cancerimmunosuppression poses a problem in the treatment of cancer.

The problem of cancer immunosuppression is solved by the presentinvention, which provides CCL22 as a novel T cell target.

Thus, the present invention provides materials and methods for treatmentof cancer diseases by targeting CCL22 expressing cancer cells directlyand by killing CCL22 expressing cancer cells. This is done by enabling Tcells to recognize the CCL22 expressing cells. Interestingly, thepresent invention discloses that cytotoxic immune responses againstCCL22 expressing cells can be raised even though CCL22 expressing cellsmay antagonize the desired effects of other immunotherapeuticapproaches. Thus, despite the fact that CCL22 enables tumor evasion byattracting immune suppressive cells such as Tregs, the present inventionshows that the immune system can react against a chemokine and that itis possible to raise a specific immune response against CCL22. Thisprovides a novel mechanism for treating e.g. cancer by inhibiting therecruitment of immune suppressive cells to the tumour andCCL22-expressing cancer cells.

Likewise, the invention provides materials and methods for treatment ofother diseases, which normally may invoke an immune response, e.g.infections. In the methods of the invention T cells are enabled to killCCL22 expressing antigen-presenting cells (APCs) and/or dendritic cells(DCs).

Thus, the invention exploits expression of the immune suppressingchemokine CCL22 in cancer cells and targets these CCL22 expressingcells.

It is an aspect of the invention to provide vaccine compositions whichsurprisingly can generate an immune response against the self-proteinCCL22.

Herein is provided a vaccine composition comprising:

-   -   a) one or more of the following:        -   (i) CCL22 of SEQ ID NO: 12 or SEQ ID NO: 15 or an            immunogenically active peptide fragment of CCL22 comprising            a consecutive sequence of amino acids of CCL22 of SEQ ID            NO:12 or SEQ ID NO: 15;        -   (ii) an immunogenically active peptide fragment of CCL22,            which is an MHC Class I-restricted peptide fragment or MHC            Class II-restricted peptide fragment;        -   (iii) a functional homologue of the polypeptides under (i)            and (ii), wherein said functional homologue shares at least            70% sequence identity with SEQ ID NO:12 or SEQ ID NO: 15,            and/or said functional homologue is an immunogenically            active polypeptide consisting of a sequence identical to a            consecutive sequence of amino acids of SEQ ID NO:1, SEQ ID            NO: 4, SEQ ID NO: 11 or SEQ ID NO: 14, except that at the            most three amino acids have been substituted, such as at the            most two amino acids, such as at the most one amino acid;        -   (iv) a polypeptide comprising any of the polypeptides under            (i), (ii) or (iii));        -   (v) a nucleic acid encoding any of the polypeptides under            (i), (ii) or (iii); and    -   b) an adjuvant        for use as a medicament.

The vaccine compositions may be used as a medicament, and may forexample be for treatment of a cancer disease where CCL22 is expressed orfor treatment of an infection causing CCL22 expression inantigen-presenting cells (APCs).

The invention also provides kit-of-parts comprising the vaccinecomposition and a second active ingredient.

The invention also describes complexes of a peptide fragment of CCL22and a Class I HLA or a Class II HLA molecule or a fragment of suchmolecule. Said peptide fragment may e.g. be any of the peptide fragmentsof CCL22 described herein below in the sections “Immunogenically activepeptide fragment of CCL22”, “Polypeptide comprising CCL22 or a fragmentthereof” and “MHC”.

The invention further discloses methods of detecting in an individualsuffering from a clinical condition the presence of CCL22 reactiveT-cells, the method comprising contacting a tumor tissue or a bloodsample with the complex comprising peptide fragments of CCL22 describedabove and detecting binding of the complex to the tissue or the bloodcells.

The invention also discloses molecules that are capable of bindingspecifically to a peptide fragment of CCL22 e.g. be any of the peptidefragments of CCL22 described herein below in the sections“Immunogenically active peptide fragment of CCL22”, “Polypeptidecomprising CCL22 or a fragment thereof” and “MHC”.

The invention also provides methods of treating a clinical conditioncharacterized by the expression of CCL22, the method comprisingadministering to an individual suffering from said clinical condition aneffective amount of the vaccine compositions of the invention, of akit-of-parts of the invention, of a composition of the invention and/orof an immunogenically active peptide fragment of CCL22, e.g. be any ofthe peptide fragments of CCL22 described herein below in the sections“Immunogenically active peptide fragment of CCL22”, “Polypeptidecomprising CCL22 or a fragment thereof” and “MHC”.

The invention also provides use of the vaccine compositions of theinvention in the manufacture of a medicament for the treatment orprevention of a clinical condition, e.g. cancer and/or inflammations.

The invention also provides methods of monitoring immunization, saidmethod comprising the steps of

-   -   a) providing a blood sample from an individual    -   b) providing an immunogenically active peptide fragment        comprising a consecutive sequence of SEQ ID NO: 12 or a        functional homologue thereof having at least 70% identity to SEQ        ID NO: 12 or a nucleic acid encoding said peptide fragment or        functional homologue;    -   c) determining whether said blood sample comprises antibodies or        T-cells comprising T-cell receptors specifically binding the        protein or peptide    -   thereby determining whether an immune response to said protein        or peptide has been raised in said individual.

Further, the invention provides immunogenically active CCL22 peptidefragments comprising a consecutive sequence of the CCL22 fragment of SEQID NO: 1, SEQ ID NO: 4 or SEQ ID NO: 11 or a functional homologuethereof, said functional homologue being a polypeptide of identicalsequence except that at the most three amino acids have beensubstituted, or a nucleic acid encoding said CCL22 peptide fragment foruse in the treatment or prevention of clinical conditions associatedwith expression of CCL22, such as cancer and/or infections.

DESCRIPTION OF DRAWINGS

FIG. 1: Action of CCL22 in cancer.

FIG. 2: CCL22 specific CD8+ T cells exist in peripheral blood.A—Intracellular staining for IFNγ and TNFα release shows that CD8+(Left) and not CD4+ (Right) T cells react against CCL22-3 peptideepitope. B—Left: CCL22-3 peptide reactive T cells can be enriched fromperipheral blood of melanoma cancer patient by stimulation with peptidepulsed autologous DCs. Right—tetramer isolated and rapidly expandedCCL22-3 specific T cell culture.

FIG. 3: CCL22 reactive T cells are cytotoxic. A—CCL22-3 but not HIVpeptide pulsed T2 cells are lysed by CCL22 specific T cells. B—CCL22longpeptide (SEQ ID NO: 1) is recognized by CCL22 specific T cells. Hence,T2 cells pulsed with CCL22long and CCL22-3 are recognized and killed. T2cells pulsed with control peptide are not recognized by CCL22-specific Tcells. C—Cytotoxic activity of CCL22 specific T cells is HLA dependent.Hence, K562 cells transfected with HLA-A2 and pulsed with CCL22-3peptide are recognized and killed. K562 cells, K562 cells pulsed withCCL22-3 or K562 cells transfected with HLA-A2 are not recognized.D—CCL22-3 peptide is recognized better than 9-mer CCL22-2 peptide.Peptide titration of CCL22-3 and CCL22-2 peptides revealed that theCCL22 specific T cells cross-react but recognize CCI22-3 with higheravidity.

FIG. 4: CCL22-reactive T cells are able to recognize and killCCL22-expressing cancer cell lines. ⁵¹Cr release assays of IFNγnon-treated or pretreated cancer cell lines: SW480—colorectaladenocarcinoma (A); MDA-MB-231—breast adenocarcinoma (B); Uke-1—acutemyeloid leukemia (C); THP-1—acute monocytic leukemia (D);RPM16666—Hodgkin's lymphoma (E); Set-2—essential thrombocytemia (F). Thesame effector CCL22-specific T cell culture was used as effector cells.G—Lysis of IFNγ induced THP-1 cells transfected with CCL22 siRNAtransfection or Mock transected by CCL22-specific T cells. Assayperformed 48 h after transfection. H—ELISA analysis of CCL22 expressionin the supernatant from siRNA transfected THP-1 cells compared to THP-1cells transfected with Mock control, 48 h after electroporation.

FIG. 5: CCL22 specific T-cell cytotoxicity is CCL22 dependent. A—CCL22specific T-cell cytotoxicity decreases with lower expression of CCL22.Thus, the lysis of CCL22 siRNA transfected THP-1 cells is lower thanthat of Mock transfected cells. B ELISA analysis of CCL22 expression insupernatant of THP-1 cells 48 h after siRNA transfection. C—CCL22specific T cells recognize dendritic cells in a CCL22 dependent manner.Hence, using flow cytometry, the release of TNF-a and CD107a wasexamined when incubating CCL22 specific T cells with autologousdendritic cells without and with transfection with CCL22 siRNA.

FIG. 6: Spontaneous CCL22-3 response peripheral blood of healthy donor.A—Example of ELISPOT response from PBMCs of a healthy donor without andwith CCL22-3 peptide as shown by IFNγ ELISPOT. B—CCL22 expression insupernatant from PBMC from the same donor after stimulation with IL-2either with CCL22-3 peptide or HIV control peptide. C—IL6 and TNFα insupernatant from PBMC from the same donor after stimulation with IL-2either with CCL22-3 peptide or HIV control peptide after peptidestimulation. D—CCL22 expression in IL-2 stimulated PMBC supernatant fromPBMC from the same donor after either CCL22-3/HLA-A2 tetramer enrichment(left) or CCL22-3/HLA-A2 tetramer depletion (right).

FIG. 7: CCL22 expression levels by ovarian cancer ASCITES cells areaffected by addition of CCL22-3 peptide. ELISA analysis of supernatantsafter 2 days (A) and 7 days (B) of cell culture of ASCITES cellsisolated from a patient with ovarian cancer stimulated with CCL22-3 orHIV peptides with the addition of IL2.

FIG. 8: CCL22₃₋₁₂-reactive T cells are present in healthy donors andcancer patients. A—IFNγ ELISPOT examples showing T-cell responsesagainst the pCCL22₃₋₁₂ epitope in three melanoma patients (MM) and twohealthy donors (HD). B—pCCL22₃₋₁₂ peptide specific IFNγ ELISPOTresponses in cancer patients and healthy donors The average number ofpCCL22₃₋₁₂-specific spots (after subtraction of spots in wells withoutadded peptide) was calculated per 3×10⁵ PBMC for each donor.C—Experimental setup of pCCL22₃₋₁₂-specific T-cell depletion/enrichmentin PBMCs. PBMCs from a healthy donor were stimulated twice withpCCL22₃₋₁₂ peptide before the pCCL22₃₋₁₂-specific T cells (anti-CCL22 Tcells) were isolated using pCCL22₃₋₁₂-PE-tetramer in combination withanti-PE magnetic beads. The remaining depleted PBMCs were divided intotwo cultures before pCCL22₃₋₁₂-tetramer isolated cells were added intoone of these resulting in a tetramer-enriched culture. CCL22-specific Tcells in a tetramer-enriched culture target CCL22 producing T cells (asindicated by black arrows). D—ELISA analysis of CCL22 in thesupernatants from pCCL22₃₋₁₂-tetramer-enriched compared totetramer-depleted PBMC cultures over time.

FIG. 9: Activation of CCL22-specific T cells decreases with CCL22 levelsin the microenvironment. A—CCL22 levels in supernatants from PBMCisolated from a healthy donor after stimulation with either pCCL22₃₋₁₂or HIV peptide as measured by CCL22 ELISA (*, P=0.01, ns—notsignificant, t-test). Experiments were performed in triplicates for eachpeptide. B—Changes in CCL22 levels in supernatants from PBMC isolatedfrom 11 healthy donor PBMCs after stimulation with pCCL22₃₋₁₂ peptidecompared to HIV control peptide as measured by CCL22 ELISA on day 7.C—Changes in CCL22 levels in in supernatants from PBMC isolated from 13cancer patients after stimulation with pCCL22₃₋₁₂ peptide compared toHIV control peptide as measured by CCL22 ELISA on day 7). Experimentswere performed in triplicates or duplicates for each peptide. D—CCL22levels in supernatants from cells isolated from ovarian cancer ascitesisolated from two ovarian cancer patients after stimulation with eitherpCCL22₃₋₁₂ or HIV peptide as measured by CCL22 ELISA.

FIG. 10: Stimulation of CCL22-specific T cells affects the PBMC cytokineprofile. A—Overall changes in IL-6 expression in supernatants from PBMCisolated from 11 cancer patients (left) or 10 healthy donors (right)after pCCL22₃₋₁₂ stimulation compared to HIV control peptide stimulation(P=0.02 and P=0.06 respectively, paired t-test). B—Overall changes inTNFα expression in supernatants from PBMC isolated from 11 cancerpatients (left) or 10 healthy donors (right) after pCCL22₃₋₁₂stimulation compared to HIV control peptide stimulation (P=0.16 andP=0.7 respectively, paired t-test).

FIG. 11: An immune response can be raised in vivo. Mice were vaccinatedby subcutaneous injections of either mCCL22long (SEQ ID NO: 13) ormCCL22short (SEQ ID NO: 14) as shown in the upper panel, once a weekover a period of three weeks, before being sacrificed one week after thelast vaccination. ELISPOT assays were performed on splenocytes preparedfrom the spleens of the sacrificed mice. To investigate IFNy response tomurine CCL22 peptides, cells were incubated with the peptides (5 μM) orR10 as control for 18-20 h at 37° C. ELISPOT was developed with mouseIFNy-specific detection Ab (R4-6A2-biotin; Mabtech) in a concentrationof 1 μg/ml in Buffer (PBS, 0.5% BSA and NaN3) for 2 h at roomtemperature, followed by 6 washes in PBS. Next, adding ofstreptavidin-ALP (1:1000; Mabtech) in Buffer for 1 h at roomtemperature. Spots were developed by adding substrate solution BCIP/NBT(Mabtech) and stopped by washing in tap water. The ELISPOTs wereanalyzed by ELISPOT Reader (CTL-Immunospot). The results show that thevaccinated mice display an increased immune response.

DEFINITIONS

Adjuvant: Any substance whose admixture with an administered immunogenicdeterminant/antigen/nucleic acid construct increases or otherwisemodifies the immune response to said determinant.

Antigen: Any substance that can bind to a clonally distributed immunereceptor (T-cell or B-cell receptor). Usually a peptide, polypeptide ora multimeric polypeptide. Antigens are preferably capable of elicitingan immune response.

APC: Antigen-presenting cell. An APC is a cell that displays antigencomplexed with MHC on its surface. T-cells may recognize this complexusing their T-cell receptor (TCR). APCs fall into two categories:professional, (of which there are three types: Dendritic cells,macrophages and B-cells) or non-professional (does not constitutivelyexpress the Major histocompatibility complex proteins required forinteraction with naive T cells; these are expressed only uponstimulation of the non-professional APC by certain cytokines such asIFN-γ).

Boost: To boost by a booster shot or dose is to administer an additionaldose of an immunizing agent, such as a vaccine, administered at a timeafter the initial dose to sustain the immune response elicited by theprevious dose of the same agent.

Carrier: Entity or compound to which antigens are coupled to aid in theinduction of an immune response.

CCL: C-C motif chemokine 22. Wild-type, human sequence of CCL22 ispresented in SEQ ID NO: 12.

CCL22_(xx-yy): As used herein this nomenclature refers to a polypeptidefragment of CCL22 consisting of amino acids xx-yy of SEQ ID NO: 12.mCCL22 refers to murine CCL22, and mCCL22_(xx-yy) refers to apolypeptide fragment of mCCL22 consisting of amino acids xx-yy of SEQ IDNO: 15.

Chimeric protein: A genetically engineered protein that is encoded by anucleotide sequence made by a splicing together of two or more completeor partial genes or a series of (non)random nucleic acids.

Clinical condition: A condition that requires medical attention, hereinespecially conditions associated with the expression of CCL22. Examplesof such conditions include: proliferative disorders, such as cancers andinfections.

Complement: A complex series of blood proteins whose action“complements” the work of antibodies. Complement destroys bacteria,produces inflammation, and regulates immune reactions.

CTL: Cytotoxic T lymphocyte. A sub group of T-cells expressing CD8 alongwith the T-cell receptor and therefore able to respond to antigenspresented by class I molecules.

Delivery vehicle: An entity whereby a nucleotide sequence or polypeptideor both can be transported from at least one media to another.

DC: Dendritic cell. (DCs) are immune cells and form part of themammalian immune system. Their main function is to process antigenmaterial and present it on the surface to other cells of the immunesystem, thus functioning as antigen-presenting cells (APCs).

Fragment: is used to indicate a non-full length part of a nucleic acidor polypeptide. Thus, a fragment is itself also a nucleic acid orpolypeptide, respectively.

Functional homologue: A functional homologue may be any polypeptide thatexhibits at least some sequence identity with a wild type version of apolypeptide and has retained at least one aspect of the originalfunctionality. Herein a functional homologue of CCL22 or an immunogenicpeptide fragment thereof is a polypeptide sharing at least some sequenceidentity with CCL22 or a fragment thereof and which has the capabilityto induce an immune response to cells expressing CCL22.

Immunogenically active peptide: Peptide capable of eliciting an immuneresponse in at least one individual after administration to saidindividual.

Individual: Generally any species or subspecies of bird, mammal, fish,amphibian, or reptile, preferably a mammal, most preferably a humanbeing.

Infection: Herein the term “infection” relates to any kind of clinicalcondition involving an invasion of the host organism by disease-causingagents. In particular, infection refers to a clinical conditioninvolving invasion of an individual by a pathogen.

Isolated: used in connection with nucleic acids, polypeptides, andantibodies disclosed herein ‘isolated’ refers to these having beenidentified and separated and/or recovered from a component of theirnatural, typically cellular, environment. Nucleic acids, polypeptides,and antibodies of the invention are preferably isolated, and vaccinesand other compositions of the invention preferably comprise isolatednucleic acids, polypeptides or isolated antibodies.

MHC: Major histocompatibility complex, two main subclasses of MHC, ClassI and Class II exist.

Nucleic acid: A chain or sequence of nucleotides that convey geneticinformation. In regards to the present invention the nucleic acid isgenerally a deoxyribonucleic acid (DNA).

Nucleic acid construct: A genetically engineered nucleic acid. Typicallycomprising several elements such as genes or fragments of same, cDNAs,promoters, enhancers, terminators, polyA tails, linkers, polylinkers,operative linkers, multiple cloning sites (MCS), markers, STOP codons,other regulatory elements, internal ribosomal entry sites (IRES) orothers.

Operative linker: A sequence of nucleotides or amino acid residues thatbind together two parts of a nucleic acid construct or (chimeric)polypeptide in a manner securing the biological processing of thenucleic acid or polypeptide.

Pathogen: a specific causative agent of disease, especially a biologicalagent such as a virus, bacteria, prion or parasite that can causedisease to its host, also referred to as an infectious agent.

PBL: Peripheral blood cells are the cellular components of blood,consisting of red blood cells, white blood cells, and platelets, whichare found within the circulating pool of blood and not sequesteredwithin the lymphatic system, spleen, liver, or bone marrow.

PBMC: A Peripheral Blood Mononuclear Cell (PBMC) is a blood cell havinga round nucleus, such as a lymphocyte or a monocyte. These blood cellsare a critical component in the immune system to fight infection andadapt to intruders. The lymphocyte population consists of T cells (CD4and CD8 positive ˜75%), B cells and NK cells (˜25% combined).

Polypeptide: Plurality of covalently linked amino acid residues defininga sequence and linked by amide bonds. The term is used analogously witholigopeptide and peptide. The term polypeptide also embracespost-translational modifications introduced by chemical orenzyme-catalyzed reactions, as are known in the art. The term can referto a variant or fragment of a polypeptide.

Pharmaceutical carriers: also termed excipients, or stabilizers arenon-toxic to the cell or individual being exposed thereto at the dosagesand concentrations employed. Often the pharmaceutical carrier is anaqueous pH buffered solution. Examples of pharmaceutical carriersinclude buffers such as phosphate, citrate, and other organic acids;antioxidants including ascorbic acid; low molecular weight (less thanabout 10 residues) polypeptide; proteins, such as serum albumin,gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, arginine or lysine; monosaccharides, disaccharides, andother carbohydrates including glucose, mannose, or dextrins; chelatingagents such as EDTA; sugar alcohols such as mannitol or sorbitol;salt-forming counterions such as sodium; and/or nonionic surfactantssuch as TWEEN™, polyethylene glycol (PEG), and PLURONICS™.

Plurality: At least two.

Proliferative disorder: Herein any preneoplastic or neoplastic disease,benign or malignant, where “neoplastic” refers to an abnormalproliferation of cells. A non-limiting example of a proliferativedisorder is cancer.

Promoter: A binding site in a DNA chain at which RNA polymerase binds toinitiate transcription of messenger RNA by one or more nearby structuralgenes.

Signal peptide: A short sequence of amino acids that determine theeventual location of a protein in the cell, also referred to as sortingpeptide.

Surfactant: A surface active agent capable of reducing the surfacetension of a liquid in which it is dissolved. A surfactant is a compoundcontaining a polar group which is hydrophilic and a non-polar groupwhich is hydrophobic and often composed of a fatty chain.

Treg: Regulatory T cells/T lymphocytes

Treatment: The term “treatment” as used herein may refer to curativetreatment and/or to ameliorating treatment and/or to treatment reducingsymptoms of disease and/or treatment delaying disease progression.

Vaccine: A substance or composition capable of inducing an immuneresponse in an individual, and particularly in a mammal, preferably in ahuman being. Also referred to as an immunogenic composition in thepresent text. A vaccine according to the present invention mayfrequently be a composition comprising at least an adjuvant and animmunogenic peptide. An immune response against an agent is a humoral,antibody and/or cellular response inducing memory in an organism,resulting in that said agent is being met by a secondary rather than aprimary response, thus reducing its impact on the host organism. Saidagent may be pathogen. In the context of the present invention the agentis preferably a cancer cell. A vaccine of the present invention may begiven as a prophylaxis, in order to reduce the risk of encountering aclinical condition and/or as a therapeutic medicament for treatment of aclinical condition. The composition may comprise one or more of thefollowing: antigen(s), nucleic acid constructs encoding one or moreantigens, carriers, adjuvants and pharmaceutical carriers.

Variant: a ‘variant’ of a given reference nucleic acid or polypeptiderefers to a nucleic acid or polypeptide that displays a certain degreeof sequence homology/identity to said reference nucleic acid orpolypeptide but is not identical to said reference nucleic acid orpolypeptide.

DETAILED DESCRIPTION OF THE INVENTION

The present disclosure relates to a vaccine composition comprising:

-   -   a) one or more of the following:        -   (i) CCL22 of SEQ ID NO: 12 or SEQ ID NO: 15 or an            immunogenically active peptide fragment of CCL22 comprising            a consecutive sequence of amino acids of CCL22 of SEQ ID NO:            12 or SEQ ID NO: 15;        -   (ii) an immunogenically active peptide fragment of CCL22,            which is an MHC Class I-restricted peptide fragment or MHC            Class II-restricted peptide fragment;        -   (iii) a functional homologue of the polypeptides under (i)            and (ii), wherein said functional homologue shares at least            70% sequence identity with SEQ ID NO: 12 or SEQ ID NO: 15,            and/or said functional homologue is an immunogenically            active polypeptide consisting of a sequence identical to a            consecutive sequence of amino acids of SEQ ID NO: 12 or SEQ            ID NO: 15, except that at the most three amino acids have            been substituted, such as at the most two amino acids, such            as at the most one amino acid;        -   (iv) a polypeptide comprising any of the polypeptides under            (i), (ii) or (iii));        -   (v) a nucleic acid encoding any of the polypeptides under            (i), (ii) or (iii); and    -   b) an adjuvant        for use as a medicament.

The disclosure also relates to a kit-of-parts comprising the vaccinecompositions described herein, and a second active ingredient.

In yet another aspect, the disclosure relates to a complex of a peptidefragment as defined herein and a Class I HLA or a Class II HLA moleculeor a fragment of such molecule.

In yet another aspect, the disclosure relates to a method of detectingin an individual suffering from a clinical condition the presence ofCCL22 reactive T-cells, the method comprising contacting a tumor tissueor a blood sample with a complex of the disclosure and detecting bindingof the complex to the tissue or the blood cells.

In yet another aspect, a molecule that is capable of bindingspecifically to a peptide fragment as defined herein is disclosed.

In yet another aspect, a molecule that is capable of blocking thebinding of the molecule capable of binding specifically to a peptidefragment as defined herein is disclosed.

In yet another aspect, a method of treating or preventing a clinicalcondition characterized by the expression of CCL22, the methodcomprising administering to an individual suffering from said clinicalcondition an effective amount of the composition, the molecule or thekit-of-parts described herein is disclosed.

In yet another aspect, the disclosure relates to the use of the vaccinecomposition, the kit-of-parts or the molecule described herein in themanufacture of a medicament for the treatment or prevention of aclinical condition.

In yet another aspect, the disclosure relates to a method of monitoringimmunization, said method comprising the steps of

-   -   a) providing a blood sample from an individual    -   b) providing CCL22 of SEQ ID NO: 12 or SEQ ID NO: 15 or an        immunogenically active peptide fragment comprising a consecutive        sequence of SEQ ID NO: 12 or SEQ ID NO: 15 or a functional        homologue thereof having at least 70% identity to SEQ ID NO: 12        or SEQ ID NO: 15 or a nucleic acid encoding said peptide        fragment or functional homologue;    -   c) determining whether said blood sample comprises antibodies or        T-cells comprising T-cell receptors specifically binding the        protein or peptide    -   thereby determining whether an immune response to said protein        or peptide has been raised in said individual.

In yet another aspect, the disclosure relates to an immunogenicallyactive CCL22 peptide fragment comprising a consecutive sequence of SEQID NO: 12 or SEQ ID NO: 15 or a functional homologue thereof, saidfunctional homologue being a polypeptide of identical sequence exceptthat at the most three amino acids have been substituted, or a nucleicacid encoding said CCL22 peptide fragment for use in the treatment orprevention of clinical conditions associated with expression of CCL22,such as cancer and/or inflammation.

Vaccine Composition

It is one of aspect of the present invention to provide a vaccinecomposition comprising one or more of the following:

-   -   (i) CCL22 of SEQ ID NO: 12 or SEQ ID NO: 15 or an        immunogenically active peptide fragment of CCL22 comprising a        consecutive sequence of amino acids of CCL22 of SEQ ID NO:12 or        SEQ ID NO: 15;    -   (ii) an immunogenically active peptide fragment of CCL22, which        is an MHC Class I-restricted peptide fragment or MHC Class        II-restricted peptide fragment;    -   (iii) a functional homologue of the polypeptides under (i) and        (ii), wherein said functional homologue shares at least 70%        sequence identity with SEQ ID NO:12 or SEQ ID NO: 15, and/or        said functional homologue is an immunogenically active        polypeptide consisting of a sequence identical to a consecutive        sequence of amino acids of SEQ ID NO:12 or SEQ ID NO: 15, except        that at the most three amino acids have been substituted, such        as at the most two amino acids, such as at the most one amino        acid;    -   (iv) a polypeptide comprising any of the polypeptides under        (i), (ii) or (iii));    -   (v) a nucleic acid encoding any of the polypeptides under        (i), (ii) or (iii).

In addition to the above-mentioned said vaccine composition preferablyalso comprises an adjuvant, which for example may be any of theadjuvants described herein below in the section “Adjuvant”.

Functional homologues, which may be used in the vaccine compositions ofthe invention are described herein below in the sections “C-C-motifchemokine 22”; “Immunogenically active peptide fragment of CCL22”;“Functional homologues” and “Polypeptides comprising CCL22 or a fragmentthereof”.

C-C Motif Chemokine 22 (CCL22)

C-C motif chemokine 22 is a protein that in humans is encoded by theCCL22 gene. The amino acid sequence of human CCL22 is set forth in SEQID NO: 12, and the sequence of murine CCL22 in SEQ ID NO: 15. Theprotein encoded by this gene is secreted by dendritic cells andmacrophages, and elicits its effects on its target cells by interactingwith cell surface chemokine receptors such as CCR4. The gene for CCL22is located in human chromosome 16 in a cluster with other chemokinescalled CX3CL1 and CCL17. CCL22 chemoattracts and recruits CD25+CD4+Tregs expressing CCR4. Expression of CCL22 has been previously shown tocause accumulation of Foxp3+ Tregs in ovarian, prostate, esophageal,gastric, and breast carcinomas.

CCL22 according to the present disclosure may be any useful CCL22.Throughout this disclosure, the term “CCL22” refers to full-lengthCCL22, such as human CCL22 as set forth in SEQ ID NO: 12 or murine CCL22as set forth in SEQ ID NO: 15. In general it is preferred that CCL22 isof the same species which it is intended to treat with the vaccinecompositions of the disclosure. In preferred embodiments of thedisclosure, the vaccine composition is intended for administration to ahuman being, and hence CCL22 may be human CCL22. The amino acid sequenceof wild type human CCL22 is presented as SEQ ID NO: 12 herein. In otherembodiments, CCL22 may be murine CCL22 as set forth in SEQ ID NO: 15.

Thus, CCL22 may preferably be CCL22 of SEQ ID NO: 12 or a functionalhomologue thereof sharing at least 70% sequence identity to CCL22 of SEQID NO: 12, and accordingly, a functional homologue preferably having atleast 75% sequence identity, for example at least 80% sequence identity,such as at least 85% sequence identity, for example at least 90%sequence identity, such as at least 91% sequence identity, for exampleat least 91% sequence identity, such as at least 92% sequence identity,for example at least 93% sequence identity, such as at least 94%sequence identity, for example at least 95% sequence identity, such asat least 96% sequence identity, for example at least 97% sequenceidentity, such as at least 98% sequence identity, for example 99%sequence identity with human CCL22 of SEQ ID NO: 12.

CCL22 may in other embodiments be CCL22 of SEQ ID NO: 15 or a functionalhomologue thereof sharing at least 70% sequence identity to CCL22 of SEQID NO: 15, and accordingly, a functional homologue preferably having atleast 75% sequence identity, for example at least 80% sequence identity,such as at least 85% sequence identity, for example at least 90%sequence identity, such as at least 91% sequence identity, for exampleat least 91% sequence identity, such as at least 92% sequence identity,for example at least 93% sequence identity, such as at least 94%sequence identity, for example at least 95% sequence identity, such asat least 96% sequence identity, for example at least 97% sequenceidentity, such as at least 98% sequence identity, for example 99%sequence identity with human CCL22 of SEQ ID NO: 15.

Functional homologues of CCL22 and methods for determining sequenceidentity are described in more detail in the section “Functionalhomologues” herein below.

Since CCL22 potentially may have unwanted activity, then in oneembodiment of the disclosure, the vaccine composition comprises mutantCCL22, which is not capable of chemoattracting and recruiting CD25+CD4+Tregs expressing CCR4, or which are only capable of doing so with anactivity of at the most 10% of the CCL22 of SEQ ID NO: 12. Such mutantCCL22 may in particular be CCL22 of SEQ ID NO: 12 wherein one or more ofthe amino acids have been mutated to another amino acid or are deleted.The mutant CCL22 may also be a mutant CCL22 fragment, for example amutant fragment of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO:11, SEQ ID NO: 13 SEQ ID NO: 14 or SEQ ID NO: 16, wherein one or more ofthe amino acids have been mutated to another amino acid or are deleted.In the context of the present invention a “functional homologue” ofCCL22 may be mutant CCL22, which does not have the catalytic activity ofwild type CCL22, but which has the capability to induce an immuneresponse to cells expressing CCL22.

Immunogenically Active Peptide Fragment of CCL22

The wild-type human CCL22, i.e. the naturally occurring non-mutatedversion of the protein is identified in SEQ ID NO: 12. The wild-typemurine CCL22 is identified in SEQ ID NO: 15. The present inventioncovers vaccine compositions comprising human or murine CCL22;immunologically active peptide fragments of human or murine CCL22;peptide fragments of human or murine CCL22, wherein at the most twoamino acids have been substituted; and/or functional homologues of Chuman or murine CL22 comprising a sequence identity of at least 70% toSEQ ID NO: 12 or SEQ ID NO: 15. The term polypeptide fragment is usedherein to define any non-full length (as compared to SEQ ID NO: 12 orSEQ ID NO:15) string of amino acid residues that are directly derivedfrom or synthesized to be identical with at least part of SEQ ID NO: 12or SEQ ID NO: 15. The peptide fragment may for example be a consecutivesequence of in the range of from 5 to 24 amino acids of SEQ ID: 12 orSEQ ID NO: 15, such as from 5 to 22 amino acids of SEQ ID NO: 12 or SEQID NO: 15, for example from 8 to 22 amino acids of SEQ ID NO: 12 or SEQID NO: 15, for example the peptide fragment may comprise SEQ ID NO: 1,SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 14or SEQ ID NO: 16.

A functional homologue can be defined as a full length or fragment ofCCL22 that differs in sequence from the wild-type CCL22, such aswild-type human or murine CCL22, but is still capable of inducing animmune response against CCL22 expressing cells such as cancer cells andDCs. The CCL22 expressed in these cells may be wild type or endogenouslymutated (such as a congenital mutant or a mutation induced during celldivision or other). A functional homologue may be a mutated version oran alternative splice variant of the wild-type CCL22. In another aspect,functional homologues of CCL22 are defined as described herein below. Afunctional homologue may be, but is not limited to, a recombinantversion of full length or fragmented CCL22 with one or more mutationsand/or one or more sequence deletions and/or additions introduced exvivo.

Accordingly, in a specific embodiment the immunogenically active peptidefragment of the invention consists of at the most 90 consecutive aminoacid residues, such as of the most 80 consecutive amino acids residues,for example at the most 70 consecutive amino acid residues, such as atthe most 60 consecutive amino acid residues, for example at the most 50consecutive amino acid residues, for example at the most 45 consecutiveamino acid residues, such as at the most 40 consecutive amino acidresidues, for example at the most 35 consecutive amino acid residues,such as at the most 30 consecutive amino acid residues, for example atthe most 25 consecutive amino acid residues, such as 18 to 25consecutive amino acids of CCL22 as identified in SEQ ID NO: 12 or SEQID NO: 15 or a functional homologue thereof; the functional homologuebeing a polypeptide of identical sequence except that at the most threeamino acids have been substituted, such as at the most two amino acidshave been substituted, such as at the most one amino acid has beensubstituted. Said immunogenically active peptide fragment may alsoconsists of at the most 80 consecutive amino acids residues, for exampleat the most 70 consecutive amino acid residues, such as at the most 60consecutive amino acid residues, for example at the most 50 consecutiveamino acid residues, for example at the most 45 consecutive amino acidresidues, such as at the most 40 consecutive amino acid residues, forexample at the most 35 consecutive amino acid residues, such as at themost 30 consecutive amino acid residues, for example at the most 25consecutive amino acid residues, such as 18 to 25 consecutive aminoacids of CCL22 as identified in SEQ ID NO: 12 or SEQ ID NO: 15, whereinone or more amino acids have been mutated to another amino acid ordeleted.

In one preferred embodiment of the invention, the immunogenically activepeptide fragment consists of in the range of 18 to 25 amino acids,preferably of 22 consecutive amino acids of CCL22 as identified in SEQID NO: 12 or SEQ ID NO: 15 or a functional homologue thereof; thefunctional homologue being a polypeptide of identical sequence exceptthat at the most three amino acids have been substituted, such as at themost two amino acids have been substituted, such as at the most oneamino acid has been substituted.

Accordingly in another specific embodiment the immunogenically activepeptide fragment of the invention consists of at the most 25 amino acidresidues, such as at the most 24 amino acid residues, such as at themost 23 amino acid residues, such as at the most 22 amino acid residues,such as at the most 21 amino acid residues, such as at the most 20 aminoacid residues, for example at the most 19 amino acid residues, such asat the most 18 amino acid residues, for example at the most 17 aminoacid residues, such as at the most 16 amino acid residues, for exampleat the most 15 amino acid residues, such as at the most 14 amino acidresidues, for example at the most 13 amino acid residues, such as at themost 12 amino acid residues, for example at the most 11 amino acidresidues, such as 8 to 10 consecutive amino acids from CCL22 of SEQ IDNO: 12 or SEQ ID NO: 15 or a functional homologue thereof; thefunctional homologue being a polypeptide of identical sequence exceptthat at the most three amino acids have been substituted, such as at themost two amino acids have been substituted, such as at the most oneamino acid has been substituted.

In one preferred embodiment of the invention, the immunogenically activepeptide comprises at the most 10 consecutive amino acid residues fromCCL22, such as at the most 9 consecutive amino acid residues, such as 8consecutive amino acid residues, such as 7 consecutive amino acidresidues from CCL22 as identified in SEQ ID NO: 12 or SEQ ID NO: 15 or afunctional homologue thereof; the functional homologue being apolypeptide of identical sequence except that at the most three aminoacids have been substituted, such as at the most two amino acids havebeen substituted, such as at the most one amino acid has beensubstituted. In particular, the immunogenically active peptide mayconsist of 10 consecutive amino acid residues from CCL22 of SEQ ID NO:12 or SEQ ID NO: 15 or the immunogenically active peptide may consist of9 consecutive amino acid residues from CCL22 of SEQ ID NO: 12 or SEQ IDNO: 15.

In another preferred embodiment of the invention, the immunogenicallyactive peptide comprises at the most 11 consecutive amino acid residuesfrom the CCL22 peptide fragment of SEQ ID NO: 1, such as at the most 10consecutive amino acid residues, such as at the most 9 consecutive aminoacid residues, such as 8 consecutive amino acid residues, such as 7consecutive amino acid residues from the CCL22 peptide fragment asidentified in SEQ ID NO: 1 or a functional homologue thereof; thefunctional homologue being a polypeptide of identical sequence exceptthat at the most three amino acids have been substituted, such as at themost two amino acids have been substituted, such as at the most oneamino acid has been substituted. In particular, the immunogenicallyactive peptide may consist of 10 consecutive amino acid residues fromthe CCL22 peptide fragment of SEQ ID NO: 1 or the immunogenically activepeptide may consist of 9 consecutive amino acid residues from the CCL22peptide fragment of SEQ ID NO: 1.

In particular, the immunogenically active peptide may consist of 10consecutive amino acid residues from the CCL22 peptide fragment of SEQID NO: 3 or the immunogenically active peptide may consist of 9consecutive amino acid residues from the CCL22 peptide fragment of SEQID NO: 3.

In another preferred embodiment of the invention, the immunogenicallyactive peptide comprises at the most 11 consecutive amino acid residuesfrom the CCL22 peptide fragment of SEQ ID NO: 4, such as at the most 10consecutive amino acid residues, such as at the most 9 consecutive aminoacid residues, such as 8 consecutive amino acid residues, such as 7consecutive amino acid residues from the CCL22 peptide fragment asidentified in SEQ ID NO: 4 or a functional homologue thereof; thefunctional homologue being a polypeptide of identical sequence exceptthat at the most three amino acids have been substituted, such as at themost two amino acids have been substituted, such as at the most oneamino acid has been substituted. In particular, the immunogenicallyactive peptide may consist of 10 consecutive amino acid residues fromthe CCL22 peptide fragment of SEQ ID NO: 4 or the immunogenically activepeptide may consist of 9 consecutive amino acid residues from the CCL22peptide fragment of SEQ ID NO: 4.

In another preferred embodiment of the invention, the immunogenicallyactive peptide comprises at the most 11 consecutive amino acid residuesfrom the CCL22 peptide fragment of SEQ ID NO: 11, such as at the most 10consecutive amino acid residues, such as at the most 9 consecutive aminoacid residues, such as 8 consecutive amino acid residues, such as 7consecutive amino acid residues from the CCL22 peptide fragment asidentified in SEQ ID NO: 11 or a functional homologue thereof; thefunctional homologue being a polypeptide of identical sequence exceptthat at the most three amino acids have been substituted, such as at themost two amino acids have been substituted, such as at the most oneamino acid has been substituted. In particular, the immunogenicallyactive peptide may consist of 10 consecutive amino acid residues fromthe CCL22 peptide fragment of SEQ ID NO: 11 or the immunogenicallyactive peptide may consist of 9 consecutive amino acid residues from theCCL22 peptide fragment of SEQ ID NO: 11.

In some embodiments, the immunogenically active peptide fragmentcomprises or consists of VXLVLLAVAY (SEQ ID NO: 16), in particular thepeptide fragment may comprise or consist of SEQ ID NO: 16, where X is avaline residue or an alanine residue and Y is a leucine residue or anisoleucine residue. Thus in some embodiments, the immunogenically activepeptide comprises at the most 11 consecutive amino acid residues whichcomprise SEQ ID NO: 16, such as at the most 10 consecutive amino acidresidues consisting of SEQ ID NO: 16.

In some embodiments of the invention the immunogenically active peptidemay be selected from the group consisting of peptides listed in Table 1or a functional homologue thereof; the functional homologue being apolypeptide of identical sequence except that at the most three aminoacids have been substituted, such as at the most two amino acids havebeen substituted, such as at the most one amino acid has beensubstituted.

In some embodiments of the invention the immunogenically active peptidemay be selected from the group consisting of peptides listed in Table 1or a functional homologue thereof, the functional homologue being apolypeptide of at least 70% sequence identity therewith, such as atleast 75% sequence identity, such as at least 80% sequence identity,such as at least 85% sequence identity, such as at least 90% sequenceidentity, such as at least 95% sequence identity, such as at least 96%sequence identity, such as at least 97% sequence identity, such as atleast 98% sequence identity, such as at least 99% sequence identitytherewith.

Accordingly, in a preferred embodiment, the immunogenically activepeptide may be the CCL22 peptide fragment of SEQ ID NO: 1 or afunctional homologue thereof, the functional homologue being apolypeptide of at least 70% sequence identity therewith, such as atleast 75% sequence identity, such as at least 80% sequence identity,such as at least 85% sequence identity, such as at least 90% sequenceidentity, such as at least 95% sequence identity, such as at least 96%sequence identity, such as at least 97% sequence identity, such as atleast 98% sequence identity, such as at least 99% sequence identitytherewith.

In another preferred embodiment, the immunogenically active peptide maybe the CCL22 peptide fragment of SEQ ID NO: 3 or a functional homologuethereof, the functional homologue being a polypeptide of at least 70%sequence identity therewith, such as at least 75% sequence identity,such as at least 80% sequence identity, such as at least 85% sequenceidentity, such as at least 90% sequence identity, such as at least 95%sequence identity, such as at least 96% sequence identity, such as atleast 97% sequence identity, such as at least 98% sequence identity,such as at least 99% sequence identity therewith.

In another preferred embodiment, the immunogenically active peptide maybe the CCL22 peptide fragment of SEQ ID NO: 4 or a functional homologuethereof, the functional homologue being a polypeptide of at least 70%sequence identity therewith, such as at least 75% sequence identity,such as at least 80% sequence identity, such as at least 85% sequenceidentity, such as at least 90% sequence identity, such as at least 95%sequence identity, such as at least 96% sequence identity, such as atleast 97% sequence identity, such as at least 98% sequence identity,such as at least 99% sequence identity therewith.

In another preferred embodiment, the immunogenically active peptide maybe the CCL22 peptide fragment of SEQ ID NO: 11 or a functional homologuethereof, the functional homologue being a polypeptide of at least 70%sequence identity therewith, such as at least 75% sequence identity,such as at least 80% sequence identity, such as at least 85% sequenceidentity, such as at least 90% sequence identity, such as at least 95%sequence identity, such as at least 96% sequence identity, such as atleast 97% sequence identity, such as at least 98% sequence identity,such as at least 99% sequence identity therewith.

In another preferred embodiment, the immunogenically active peptide maybe the CCL22 peptide fragment of SEQ ID NO: 13 or a functional homologuethereof, the functional homologue being a polypeptide of at least 70%sequence identity therewith, such as at least 75% sequence identity,such as at least 80% sequence identity, such as at least 85% sequenceidentity, such as at least 90% sequence identity, such as at least 95%sequence identity, such as at least 96% sequence identity, such as atleast 97% sequence identity, such as at least 98% sequence identity,such as at least 99% sequence identity therewith.

In another preferred embodiment, the immunogenically active peptide maybe the CCL22 peptide fragment of SEQ ID NO: 14 or a functional homologuethereof, the functional homologue being a polypeptide of at least 70%sequence identity therewith, such as at least 75% sequence identity,such as at least 80% sequence identity, such as at least 85% sequenceidentity, such as at least 90% sequence identity, such as at least 95%sequence identity, such as at least 96% sequence identity, such as atleast 97% sequence identity, such as at least 98% sequence identity,such as at least 99% sequence identity therewith.

In another preferred embodiment, the immunogenically active peptide maybe the CCL22 peptide fragment of SEQ ID NO: 16 or a functional homologuethereof, the functional homologue being a polypeptide of at least 70%sequence identity therewith, such as at least 75% sequence identity,such as at least 80% sequence identity, such as at least 85% sequenceidentity, such as at least 90% sequence identity, such as at least 95%sequence identity, such as at least 96% sequence identity, such as atleast 97% sequence identity, such as at least 98% sequence identity,such as at least 99% sequence identity therewith.

TABLE 1 Useful CCL22 peptides Amino acid numbers in SEQ ID NO NameSEQ ID NO: 1 Sequence SEQ ID NO: 1 CCL22LONG CCL22₁₋₂₂MDRLQTALLVVLVLLAVALQAT SEQ ID NO: 2 CCL22-1 CCL22₇₋₁₅ ALLVVLVLLSEQ ID NO: 3 CCL22-2 CCL22₃₋₁₁ RLQTALLVV SEQ ID NO: 4 CCL22-3 CCL22₃₋₁₂RLQTALLVVL SEQ ID NO: 5 CCL22-4 CCL22₁₁₋₁₉ VLVLLAVAL SEQ ID NO: 6CCL22-5 CCL22₅₋₁₃ QTALLVVLV SEQ ID NO: 7 CCL22-6 CCL22₁₄₋₂₂ LLAVALQATSEQ ID NO: 8 CCL22-7 CCL22₈₋₁₇ LLVVLVLLAV SEQ ID NO: 9 CCL22-8 CCL22₉₋₁₇LVVLVLLAV SEQ ID NO: 10 CCL22-9 CCL22₆₋₁₅ TALLVVLVL SEQ ID NO: 11CCL22SIGNAL CCL22₁₋₂₄ MDRLQTALLVVLVL LAVALQATEA Amino acid numbers inSEQ ID NO Name SEQ ID NO: 15 Sequence SEQ ID NO: 13 mCCL22longmCCL22₁₋₂₂ MATLRVPLLVALVLLAVAIQTS SEQ ID NO: 14 mCCL22short mCCL22₁₀₋₁₉VALVLLAVAI SEQ ID NO: 16 Consensus VXLVLLAVAY sequence

In a preferred embodiment of the invention the immunogenically activepeptide is selected from the group consisting of:

-   a) SEQ ID NO: 1 (CCL22₁₋₂₂);-   b) SEQ ID NO:3 (CCL22₃₋₁₁);-   c) SEQ ID NO: 4 (CCL22₃₋₁₂);-   d) SEQ ID NO: 11 (CCL22₁₋₂₄);-   e) SEQ ID NO: 14 (mCCL22₁₀₋₁₉) and-   f) a functional homologue of the polypeptide according to any of a)    to d); the functional homologue being a polypeptide of identical    sequence except that at the most three amino acids have been    substituted, such as at the most two amino acids have been    substituted, such as at the most one amino acid has been    substituted.

Other peptides of the invention comprise (or more preferably consist of)between 4 and 90, preferably between 5 and 80, more preferably between10 and 70, yet more preferably between 12 and 60, even more preferablybetween 15 and 40, such as between 18 and 25 contiguous amino acids ofCCL22 of SEQ ID NO: 12 or SEQ ID NO: 15 or a functional homologuethereof having at least 70%, preferably at least 80%, more preferably atleast 90%, even more preferably at least 95%, yet more preferably atleast 98%, for example at least 99% sequence identity to SEQ ID NO: 12or SEQ ID NO: 15.

Functional Homologues

Functional homologues of CCL22 or immunogenically active fragmentsthereof, are polypeptides, which also are immunogenically active, andwhich shares at least some degree of sequence identity with CCL22, andin particular with CCL22 of SEQ ID NO: 12 or SEQ ID NO: 15.

For shorter polypeptides, such as for polypeptide shorter than 50 aminoacids, for example shorter than 25 amino acids, then functionalhomologue may be an immunogenically active polypeptide of identicalsequence except that at the most three amino acids have beensubstituted, such as at the most two amino acids have been substituted,such as at the most one amino acid has been substituted.

Alternatively, a functional homologue may be an immunogenically activepolypeptide sharing at least 70% sequence identity to CCL22 of SEQ IDNO: 12 or SEQ ID NO: 15, and accordingly, a functional homologuepreferably has at least 75% sequence identity, for example at least 80%sequence identity, such as at least 85% sequence identity, for exampleat least 90% sequence identity, such as at least 91% sequence identity,for example at least 91% sequence identity, such as at least 92%sequence identity, for example at least 93% sequence identity, such asat least 94% sequence identity, for example at least 95% sequenceidentity, such as at least 96% sequence identity, for example at least97% sequence identity, such as at least 98% sequence identity, forexample 99% sequence identity with human CCL22 of SEQ ID NO: 12 or SEQID NO: 15.

In a preferred embodiment, a functional homologue is an immunogenicallyactive polypeptide sharing at least 70% sequence identity to the CCL22peptide fragment of SEQ ID NO: 1, and accordingly, a functionalhomologue preferably has at least 75% sequence identity, for example atleast 80% sequence identity, such as at least 85% sequence identity, forexample at least 90% sequence identity, such as at least 91% sequenceidentity, for example at least 91% sequence identity, such as at least92% sequence identity, for example at least 93% sequence identity, suchas at least 94% sequence identity, for example at least 95% sequenceidentity, such as at least 96% sequence identity, for example at least97% sequence identity, such as at least 98% sequence identity, forexample 99% sequence identity with the CCL22 peptide fragment of SEQ IDNO: 1.

In a preferred embodiment, a functional homologue is an immunogenicallyactive polypeptide sharing at least 70% sequence identity to the CCL22peptide fragment of SEQ ID NO: 3, and accordingly, a functionalhomologue preferably has at least 75% sequence identity, for example atleast 80% sequence identity, such as at least 85% sequence identity, forexample at least 90% sequence identity, such as at least 91% sequenceidentity, for example at least 91% sequence identity, such as at least92% sequence identity, for example at least 93% sequence identity, suchas at least 94% sequence identity, for example at least 95% sequenceidentity, such as at least 96% sequence identity, for example at least97% sequence identity, such as at least 98% sequence identity, forexample 99% sequence identity with the CCL22 peptide fragment of SEQ IDNO: 3.

In another preferred embodiment, a functional homologue is animmunogenically active polypeptide sharing at least 70% sequenceidentity to the CCL22 peptide fragment of SEQ ID NO: 4, and accordingly,a functional homologue preferably has at least 75% sequence identity,for example at least 80% sequence identity, such as at least 85%sequence identity, for example at least 90% sequence identity, such asat least 91% sequence identity, for example at least 91% sequenceidentity, such as at least 92% sequence identity, for example at least93% sequence identity, such as at least 94% sequence identity, forexample at least 95% sequence identity, such as at least 96% sequenceidentity, for example at least 97% sequence identity, such as at least98% sequence identity, for example 99% sequence identity with the CCL22peptide fragment of SEQ ID NO: 4.

In another preferred embodiment, a functional homologue is animmunogenically active polypeptide sharing at least 70% sequenceidentity to the CCL22 peptide fragment of SEQ ID NO: 11, andaccordingly, a functional homologue preferably has at least 75% sequenceidentity, for example at least 80% sequence identity, such as at least85% sequence identity, for example at least 90% sequence identity, suchas at least 91% sequence identity, for example at least 91% sequenceidentity, such as at least 92% sequence identity, for example at least93% sequence identity, such as at least 94% sequence identity, forexample at least 95% sequence identity, such as at least 96% sequenceidentity, for example at least 97% sequence identity, such as at least98% sequence identity, for example 99% sequence identity with the CCL22peptide fragment of SEQ ID NO: 11.

In another preferred embodiment, a functional homologue is animmunogenically active polypeptide sharing at least 70% sequenceidentity to the CCL22 peptide fragment of SEQ ID NO: 13, andaccordingly, a functional homologue preferably has at least 75% sequenceidentity, for example at least 80% sequence identity, such as at least85% sequence identity, for example at least 90% sequence identity, suchas at least 91% sequence identity, for example at least 91% sequenceidentity, such as at least 92% sequence identity, for example at least93% sequence identity, such as at least 94% sequence identity, forexample at least 95% sequence identity, such as at least 96% sequenceidentity, for example at least 97% sequence identity, such as at least98% sequence identity, for example 99% sequence identity with the CCL22peptide fragment of SEQ ID NO: 13.

In another preferred embodiment, a functional homologue is animmunogenically active polypeptide sharing at least 70% sequenceidentity to the CCL22 peptide fragment of SEQ ID NO: 14, andaccordingly, a functional homologue preferably has at least 75% sequenceidentity, for example at least 80% sequence identity, such as at least85% sequence identity, for example at least 90% sequence identity, suchas at least 91% sequence identity, for example at least 91% sequenceidentity, such as at least 92% sequence identity, for example at least93% sequence identity, such as at least 94% sequence identity, forexample at least 95% sequence identity, such as at least 96% sequenceidentity, for example at least 97% sequence identity, such as at least98% sequence identity, for example 99% sequence identity with the CCL22peptide fragment of SEQ ID NO: 14.

In another preferred embodiment, a functional homologue is animmunogenically active polypeptide sharing at least 70% sequenceidentity to the CCL22 peptide fragment of SEQ ID NO: 16, andaccordingly, a functional homologue preferably has at least 75% sequenceidentity, for example at least 80% sequence identity, such as at least85% sequence identity, for example at least 90% sequence identity, suchas at least 91% sequence identity, for example at least 91% sequenceidentity, such as at least 92% sequence identity, for example at least93% sequence identity, such as at least 94% sequence identity, forexample at least 95% sequence identity, such as at least 96% sequenceidentity, for example at least 97% sequence identity, such as at least98% sequence identity, for example 99% sequence identity with the CCL22peptide fragment of SEQ ID NO: 16.

In some embodiments, a functional homologue is an immunogenically activepolypeptide differing from the CCL22 peptide fragment of SEQ ID NO: 1 byat least one amino acid, such as at least two amino acids, such as atleast three amino acids.

In some embodiments, a functional homologue is an immunogenically activepolypeptide differing from the CCL22 peptide fragment of SEQ ID NO: 3 byat least one amino acid, such as at least two amino acids, such as atleast three amino acids.

In another embodiment, a functional homologue is an immunogenicallyactive polypeptide differing from the CCL22 peptide fragment of SEQ IDNO: 4 by at least one amino acid, such as at least two amino acids, suchas at least three amino acids.

In another embodiment, a functional homologue is an immunogenicallyactive polypeptide differing from the CCL22 peptide fragment of SEQ IDNO: 11 by at least one amino acid, such as at least two amino acids,such as at least three amino acids.

In some embodiments, a functional homologue is an immunogenically activepolypeptide differing from the CCL22 peptide fragment of SEQ ID NO: 14by at least one amino acid, such as at least two amino acids, such as atleast three amino acids.

In some embodiments, a functional homologue is an immunogenically activepolypeptide differing from the CCL22 peptide fragment of SEQ ID NO: 14by at least one amino acid, such as at least two amino acids, such as atleast three amino acids.

In some embodiments, a functional homologue is an immunogenically activepolypeptide differing from the CCL22 peptide fragment of SEQ ID NO: 16by at least one amino acid, such as at least two amino acids, such as atleast three amino acids.

Sequence identity can be calculated using a number of well-knownalgorithms and applying a number of different gap penalties. Thesequence identity is calculated relative to full-length referencesequence, e.g. to full length SEQ ID NO: 1. Any sequence alignment tool,such as but not limited to FASTA, BLAST, or LALIGN may be used forsearching homologues and calculating sequence identity. Moreover, whenappropriate any commonly known substitution matrix, such as but notlimited to PAM, BLOSSUM or PSSM matrices may be applied with the searchalgorithm. For example, a PSSM (position specific scoring matrix) may beapplied via the PSI-BLAST program. Moreover, sequence alignments may beperformed using a range of penalties for gap opening and extension. Forexample, the BLAST algorithm may be used with a gap opening penalty inthe range 5-12, and a gap extension penalty in the range 1-2.

Functional homologues may further comprise chemical modifications suchas ubiquitination, labeling (e.g., with radionuclides, various enzymes,etc.), pegylation (derivatization with polyethylene glycol), or byinsertion (or substitution by chemical synthesis) of amino acids (aminoacids) such as ornithine, which do not normally occur in human proteins,however it is preferred that the functional equivalent does not containchemical modifications.

Any changes made to the sequence of amino acid residues compared to thatof CCL22 of SEQ ID NO: 12 or SEQ ID NO: 15, or compared to the CCL22fragments of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 11,SEQ ID NO: 13 or SEQ ID NO: 14 are preferably conservativesubstitutions. A person skilled in the art will know how to make andassess ‘conservative’ amino acid substitutions, by which one amino acidis substituted for another with one or more shared chemical and/orphysical characteristics. Conservative amino acid substitutions are lesslikely to affect the functionality of the protein. Amino acids may begrouped according to shared characteristics. A conservative amino acidsubstitution is a substitution of one amino acid within a predeterminedgroup of amino acids for another amino acid within the same group,wherein the amino acids within a predetermined groups exhibit similar orsubstantially similar characteristics.

Thus, in an embodiment of the present invention, the vaccine compositioncomprises a polypeptide consisting of a consecutive sequence of CCL22 ofSEQ ID NO: 12 or SEQ ID NO: 15 in the range of 8 to 50 amino acids,preferably in the range of 8 to 10 or 20 to 25 amino acids, wherein atthe most three amino acids have been substituted, and where thesubstitution preferably is conservative.

Polypeptides Comprising CCL22 or a Fragment Thereof

It is also comprised within the invention that the vaccine compositionsof the invention may comprise a polypeptide comprising either CCL22 or afragment thereof. Thus, the immunogenically active peptide fragment ofCCL22 may be a polypeptide comprising a CCL22 fragment, for example anyof the polypeptides described herein in this section.

In particular, such polypeptides may comprise full length CCL22, such asany of the CCL22s described herein above in the section “C-C motifchemokine 22”. For example the polypeptide may comprise CCL22 of SEQ IDNO: 12 or SEQ ID NO: 15 or a functional homologue thereof sharing atleast 70%, such as at least 80%, for example at least 90%, such as atleast 95% sequence identity therewith. In particular, such polypeptidesmay comprise at the most 90, such as at the most 50, for example at themost 25, such as at the most 10 amino acids in addition to CCL22 of SEQID NO: 12 or SEQ ID NO: 15.

It is also comprised within the invention that the vaccine compositionsmay comprise a polypeptide comprising a fragment of CCL22, such as anyof the fragments described herein above in the section “Immunogenicallyactive peptide fragment of CCL22”. Said polypeptide may also compriseany of the immunogenically active peptide fragments of CCL22, which isan MHC Class I-restricted peptide fragment or MHC Class II-restrictedpeptide fragment, such as any of the an MHC Class I-restricted peptidefragments or MHC Class II-restricted peptide fragments described in thesection “MHC”.

Thus, said polypeptide may be a polypeptide of at the most 400 aminoacids, such as at the most 300 amino acids, for example at the most 200amino acids, such as at the most 100 amino acids, for example at themost 50 amino acids comprising a consecutive sequence of amino acids ofSEQ ID NO: 12 or SEQ ID NO: 15, wherein said consecutive sequence ofamino acids of SEQ ID NO:12 or SEQ ID NO: 15 consists of at the most 50amino acid residues, for example at the most 45 amino acid residues,such as at the most 40 amino acid residues, for example at the most 35amino acid residues, such as at the most 30 amino acid residues, forexample at the most 25 amino acid residues, such as in the range of 18to 25, such as in the range of 8 to 10 consecutive amino acids fromCCL22 of SEQ ID NO: 12 or SEQ ID NO: 15 or a functional homologuethereof.

In particular, said polypeptide may be a polypeptide of at the most 100consecutive amino acid residues, such as at the most 90 consecutiveamino acid residues, such as at the most 80 consecutive amino acidresidues, for example at the most 70 consecutive amino acid residues,such as at the most 60 consecutive amino acid residues, for example atthe most 50 consecutive amino acid residues, for example at the most 45consecutive amino acid residues, such as at the most 40 consecutiveamino acid residues, for example at the most 35 consecutive amino acidresidues, such as at the most 30 consecutive amino acid residues, forexample at the most 25 consecutive amino acid residues, such as 18 to 25consecutive amino acid residues, such as of 20 consecutive amino acidsof CCL22 as identified in SEQ ID NO: 12 or SEQ ID NO: 15 or a functionalhomologue thereof, and comprising an immunogenically active peptideselected from the group consisting of:

-   a) SEQ ID NO: 1 (CCL22₁₋₂₂);-   b) SEQ ID NO:3 (CCL22₃₋₁₁);-   c) SEQ ID NO: 4 (CCL22₃₋₁₂);-   d) SEQ ID NO: 11 (CCL22₁₋₂₄);-   e) SEQ ID NO: 14 (mCCL22₁₀₋₁₉);-   f) any of the sequences mentioned in Table 1; and-   g) a functional homologue of the polypeptide according to any of a)    to d); the functional homologue being a polypeptide of identical    sequence except that at the most three amino acids have been    substituted, such as at the most two amino acids have been    substituted, such as at the most one amino acid has been    substituted.

In some embodiments, the immunogenically active peptide comprises asequence as set forth in SEQ ID NO: 16, i.e. has the sequence VXLVLLAVAY(SEQ ID NO: 16), where X is selected from the group consisting of valineand alanine, and Y is selected from the group consisting of isoleucineand leucine.

Said polypeptide may also be a polypeptide of at the most 100 aminoacids, such as at the most 50 amino acids, for example at the most 30amino acids, such as at the most 20 amino acids, for example at the most15 amino acids comprising a consecutive sequence of amino acids of SEQID NO: 12 or SEQ ID NO: 15, wherein said consecutive sequence of aminoacids of SEQ ID NO: 12 or SEQ ID NO: 15 consists of in the range of 8 to10, such as of 9 or 10 consecutive amino acids from CCL22 of SEQ ID NO:12 or SEQ ID NO: 15 or a functional homologue thereof. Thus, saidpolypeptide may be a polypeptide of at the most 100 amino acids, such asat the most 50 amino acids, for example at the most 30 amino acids, suchas at the most 20 amino acids, for example at the most 15 amino acidscomprising an immunogenically active peptide selected from the groupconsisting of:

-   a) SEQ ID NO: 1 (CCL22₁₋₂₂);-   b) SEQ ID NO: 3 (CCL22₃₋₁₁);-   c) SEQ ID NO: 4 (CCL22₃₋₁₂);-   d) SEQ ID NO: 11 (CCL22₁₋₂₄);-   e) SEQ ID NO: 14 (mCCL22₁₀₋₁₉)-   f) any of the sequences mentioned in Table 1; and-   g) a functional homologue of the polypeptide according to any of a)    to c); the functional homologue being a polypeptide of identical    sequence except that at the most three amino acids have been    substituted, such as at the most two amino acids have been    substituted, such as at the most one amino acid has been    substituted.

In some embodiments, the immunogenically active peptide consist of atthe most 100 amino acids, such as at the most 50 amino acids, forexample at the most 30 amino acids, such as at the most 20 amino acids,for example at the most 15 amino acids comprising the sequence of aminoacids of VXLVLLAVAY (SEQ ID NO: 16). In particular, said polypeptide maybe a polypeptide of at the most 100 consecutive amino acid residues,such as at the most 90 consecutive amino acid residues, such as at themost 80 consecutive amino acid residues, for example at the most 70consecutive amino acid residues, such as at the most 60 consecutiveamino acid residues, for example at the most 50 consecutive amino acidresidues, for example at the most 45 consecutive amino acid residues,such as at the most 40 consecutive amino acid residues, for example atthe most 35 consecutive amino acid residues, such as at the most 30consecutive amino acid residues, for example at the most 25 consecutiveamino acid residues, such as 18 to 25 consecutive amino acid residues,such as of 20 consecutive amino acids of CCL22 as identified in SEQ IDNO: 12 or SEQ ID NO: 15 or a functional homologue thereof, wherein saidconsecutive sequence comprises VXLVLLAVAY (SEQ ID NO: 16). It ispreferred that within SEQ ID NO:16, X is selected from the groupconsisting of valine and alanine, and Y is selected from the groupconsisting of isoleucine and leucine

MHC

It is comprised within the invention that the immunogenically activepeptide fragments of CCL22 may be an MHC Class I-restricted peptidefragment or MHC Class II-restricted peptide fragment, such as any of thean MHC Class I-restricted peptide fragments or MHC Class II-restrictedpeptide fragments described in this section.

There are two types of MHC molecules; MHC class I molecules and MHCclass II molecules. MHC class I molecules are recognized by CD8 T-cells,which are the principal effector cells of the adaptive immune response.MHC class II molecules are mainly expressed on the surface of antigenpresenting cells (APCs), the most important of which appears to be thedendritic cells. APCs stimulate naïve T-cells, as well as other cells inthe immune system. They stimulate both CD8 T-cells and CD4 T-cells.

In one embodiment, the invention provides immunogenically active CCL22peptides (optionally comprised in larger peptides and/or in vaccinecompositions), wherein said immunogenically active CCL2 peptides are MHCClass I-restricted peptide fragments consisting of 8-10 consecutiveamino acids from CCL22 of SEQ ID NO: 12 or SEQ ID NO: 15, such as thepeptide fragment of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO:11, SEQ ID NO: 13 or SEQ ID NO: 14 or a functional homologue thereof,wherein at the most two amino acids of SEQ ID NO: 12 or SEQ ID NO: 1,SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 11 or SEQ ID NO: 13 or SEQ IDNO: 14 or SEQ ID NO: 16 have been substituted, which are characterizedby having at least one of several features, one of which is the abilityto bind to the Class I HLA molecule to which it is restricted at anaffinity as measured by the amount of the peptide that is capable ofhalf maximal recovery of the Class I HLA molecule (C₅₀ value) which isat the most 50 μM as determined by the assembly binding assay asdescribed herein. This assembly assay is based on stabilization of theHLA molecule after loading of peptide to the peptide transporterdeficient cell line T2. Subsequently, correctly folded stable HLA heavychains are immunoprecipitated using conformation dependent antibodiesand the peptide binding is quantitated. The peptides of this embodimentcomprises (or more preferably consists of) at the most 100, preferablyat the most 50, more preferably at the most 25, yet more preferably atthe most 20, yet even more preferably at the most 15, such as at themost 10, for example in the range of 8 to 10 consecutive amino acids ofCCL22 of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 11, SEQ IDNO: 13 or SEQ ID NO: 14 or a functional homologue thereof wherein at themost two amino acids of SEQ ID NO: 12 or SEQ ID NO: 15 have beensubstituted.

This assay provides a simple means of screening candidate peptides fortheir ability to bind to a given HLA allele molecule at the aboveaffinity. In preferred embodiments, the peptide fragment of theinvention in one having a C₅₀ value, which is at the most 30 μM, such asa C₅₀ value, which is at the most 20 μM including C₅₀ values of at themost 10 μM, at the most 5 μM and at the most 2 μM.

In another preferred embodiment, there are provided novel MHC ClassII-restricted peptide fragments of CCL22 of SEQ ID NO: 12 or SEQ ID NO:15, such as the peptides of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4,SEQ ID NO: 11, SEQ ID NO: 13 or SEQ ID NO: 14 or functional homologuesthereof, wherein at the most two amino acids of SEQ ID NO: 12 or SEQ IDNO: 15 have been substituted (also referred to herein as “peptides”),which are characterized by having at least one of several featuresdescribed herein below. The peptides of this embodiment comprise (ormore preferably consist of) between 4 and 93, preferably between 8 and90, more preferably between 10 and 75, yet more preferably between 12and 60, even more preferably between 15 and 40, such as between 18 and25 consecutive amino acids of CCL22 of SEQ ID NO: 12 or SEQ ID NO: 15 ora functional homologue thereof, wherein at the most two, preferably atthe most one amino acids of SEQ ID NO: 12 or SEQ ID NO: 15 have beensubstituted. In a preferred embodiment, the peptides comprise (or morepreferably consist of) between 4 and 15, preferably between 5 and 14,more preferably between 6 and 13, yet more preferably between 7 and 12,even more preferably between 8 and 11, such as between 8 and 10, such as9 consecutive amino acids of the CCL22 peptide fragment of SEQ ID NO: 1.In another preferred embodiment, the peptides comprise (or morepreferably consist of) between 4 and 9, preferably between 5 and 9, morepreferably between 6 and 9, yet more preferably between 7 and 8, such as8 or 9 consecutive amino acids of the CCL22 peptide fragment of SEQ IDNO: 3. In another preferred embodiment, the peptides comprise (or morepreferably consist of) between 4 and 10, preferably between 5 and 10,more preferably between 6 and 10, yet more preferably between 7 and 9,such as 9 or 10 consecutive amino acids of the CCL22 peptide fragment ofSEQ ID NO: 4. In yet another preferred embodiment, the peptides comprise(or more preferably consist of) between 4 and 15, preferably between 5and 14, more preferably between 6 and 13, yet more preferably between 7and 12, even more preferably between 8 and 11, such as between 8 and 10,such as 9 consecutive amino acids of the CCL22 peptide fragment of SEQID NO: 11. In a preferred embodiment, the peptides comprise (or morepreferably consist of) between 4 and 15, preferably between 5 and 14,more preferably between 6 and 13, yet more preferably between 7 and 12,even more preferably between 8 and 11, such as between 8 and 10, such as9 consecutive amino acids of the CCL22 peptide fragment of SEQ ID NO:13. In yet another preferred embodiment, the peptides comprise (or morepreferably consist of) between 4 and 10, preferably between 5 and 10,more preferably between 6 and 10, yet more preferably between 7 and 10,such as 8 or 9 consecutive amino acids of the CCL22 peptide fragment ofSEQ ID NO: 14. In yet another preferred embodiment, the peptidescomprise (or more preferably consist of) between 4 and 10, preferablybetween 5 and 10, more preferably between 6 and 10, yet more preferablybetween 7 and 10, such as 8 or 9 consecutive amino acids of the CCL22peptide fragment of SEQ ID NO: 16.

Thus there are provided novel MHC Class I-restricted peptide fragmentsof 8-10 amino acids or novel MHC Class II-restricted peptide fragmentsof 18-25 amino acids of CCL22 of SEQ ID NO: 12 or SEQ ID NO: 15 or afunctional homologue thereof, wherein at the most two amino acids of SEQID NO: 12 or SEQ ID NO: 15 have been substituted, which arecharacterized by having at least one of several features describedherein below, one of which is the ability to bind to the Class I orClass II HLA molecule to which it is restricted.

In particular embodiments there is provided a peptide fragment, which isan MHC Class I-restricted peptide or an MHC class II-restricted peptidehaving at least one of the following characteristics:

(i) capable of eliciting INF-γ-producing cells in a PBL population of atleast one cancer patient at a frequency of at least 1 per 10⁴ PBLs asdetermined by an ELISPOT assay, and/or

(ii) capable of in situ detection in a tumor tissue of CTLs that arereactive with the epitope peptide.

(iii) capable of inducing the growth of CCL22 specific T-cells in vitro.

More preferred peptides according to the present invention are peptidescapable of raising a specific T-cell response as determined by anELISPOT assay, for example the ELISPOT assay described in Example 1herein below. Some peptides although they do not bind MHC class I orclass II with high affinity, may still give rise to a T-cell response asdetermined by ELISPOT. Other peptides capable of binding MHC class I orclass II with high affinity also give rise to a T-cell response asdetermined by ELISPOT. Both kinds of peptides are preferred peptidesaccording to the invention.

Hence, preferred peptides according to the present invention arepeptides capable of raising a specific T-cell response as measured by anELISPOT assay, wherein more than 50 peptide specific spots per 10⁸cells, more preferably per 10⁷, even more preferably per 10⁶, yet morepreferably per 10⁵ cells, such as per 10⁴ cells are measured.

Most preferred peptides according to the present invention are peptidesthat are capable of eliciting a cellular immune response in anindividual suffering from a clinical condition characterized by theexpression of CCL22, the clinical condition preferably being a cancer orinfection, and most preferably a cancer.

As described above, the HLA system represents the human majorhistocompatibility (MHC) system. Generally, MHC systems control a rangeof characteristics: transplantation antigens, thymus dependent immuneresponses, certain complement factors and predisposition for certaindiseases. More specifically, the MHC codes for three different types ofmolecules, i.e. Class I, II and III molecules, which determine the moregeneral characteristics of the MHC. Of these molecules, the Class Imolecules are so-called HLA-A, HLA-B and HLA-C molecules that arepresented on the surface of most nucleated cells and thrombocytes.

The peptides of the present invention are characterized by their abilityto bind to (being restricted by) a particular MHC Class I HLA molecule.Thus, in one embodiment the peptide is one which is restricted by a MHCClass I HLA-A molecule including HLA-A1, HLA-A2, HLA-A3, HLA-A9,HLA-A10, HLA-A11, HLA-Aw19, HLA-A23(9), HLA-A24(9), HLA-A25(10),HLA-A26(10), HLA-A28, HLA-A29(w19), HLA-A30(w19), HLA-A31(w19),HLA-A32(w19), HLA-Aw33(w19), HLA-Aw34(10), HLA-Aw36, HLA-Aw43,HLA-Aw66(10), HLA-Aw68(28), HLA-A69(28). More simple designations arealso used throughout the literature, where only the primary numericdesignation is used, e.g. HLA-A19 or HLA-A24 instead of HLA-Aw19 andHLA-A24(49), respectively. In specific embodiments, the peptide of theinvention is restricted a MHC Class I HLA species selected from thegroup consisting of HLA-A1, HLA-A2, HLA-A3, HLA-A11 and HLA-A24. Inspecific embodiment, the peptide of the invention is restricted a MHCClass I HLA species HLA-A2 or HLA-A3.

In further useful embodiments, the peptide of the invention is apeptide, which is restricted by a MHC Class I HLA-B molecule includingany of the following: HLA-B5, HLA-B7, HLA-B8, HLA-B12, HLA-B13, HLA-B14,HLA-B15, HLA-B16, HLA-B17, HLA-B18, HLA-B21, HLA-Bw22, HLA-B27, HLA-B35,HLA-B37, HLA-B38, HLA-B39, HLA-B40, HLA-Bw41, HLA-Bw42, HLA-B44,HLA-B45, HLA-Bw46 and HLA-Bw47. In specific embodiments of theinvention, the MHC Class I HLA-B species to which the peptide of theinvention is capable of binding is selected from HLA-B7, HLA-B35,HLA-B44, HLA-B8, HLA-B15, HLA-B27 and HLA-B51.

In further useful embodiments, the peptide of the invention is apeptide, which is restricted by a MHC Class I HLA-C molecule includingbut not limited to any of the following: HLA-Cw1, HLA-Cw2, HLA-Cw3,HLA-Cw4, HLA-Cw5, HLA-Cw6, HLA-Cw7 and HLA-Cw1.

In further useful embodiments, the peptide of the invention is apeptide, which is restricted by a MHC Class II HLA molecule includingbut not limited to any of the following: HLA-DPA-1, HLA-DPB-1, HLA-DQA1,HLA-DQB1, HLA-DRA, HLA-DRB and all alleles in these groups and HLA-DM,HLA-DO.

The selection of peptides potentially having the ability to bind to aparticular HLA molecule can be made by the alignment of known sequencesthat bind to a given particular HLA molecule to thereby reveal thepredominance of a few related amino acids at particular positions in thepeptides. Such predominant amino acid residues are also referred toherein as “anchor residues” or “anchor residue motifs”. By followingsuch a relatively simple procedure based on known sequence data that canbe found in accessible databases, peptides can be derived from CCL22,which are likely to bind to a specific HLA molecule. Representativeexamples of such analyses for a range of HLA molecules are given in thebelow table:

TABLE 2 HLA allele Position 1 Position 2 Position 3 Position 5 Position6 Position 7 C-terminal HLA-A1 T, S D, E L Y HLA-A2 L, M V L, V HLA-A3L, V, M F, Y K, Y, F HLA-A11 V, I, F, Y M, L, F, Y, I K, R HLA-A23 I, YW, I HLA-A24 Y I, V F I, L, F HLA-A25 M, A, T I W HLA-A26 E, D V, T, I,L, F I, L, V Y, F HLA-A28 E, D V, A, L A, R HLA-A29 E Y, L HLA-A30 Y, L,F, V Y HLA-A31 L, M, F, Y R HLA-A32 I, L W HLA-A33 Y, I, L, V R HLA-A34V, L R HLA-A66 E, D T, V R, K HLA-A68 E, D T, V R, K HLA-A69 V, T, A V,L HLA-A74 T V, L HLA-B5 A, P F, Y I, L HLA-B7 P L, F HLA-B8 K K, R LHLA-B14 R, K L, V HLA-B15 Q, L, K, P, F, Y, W (B62) H, V, I, M, S, THLA-B17 L, V HLA-B27 R Y, K, F, L HLA-B35 P I, L, M, Y HLA-B37 D, E I,L, M HLA-B38 H D, E F, L HLA-B39 R, H L, F HLA-B40 E F, I, V L, V, A, W,(B60, 61) M, T, R HLA-B42 L, P Y, L HLA-B44 E F, Y, W HLA-B46 M, I, L, VY, F HLA-B48 Q, K L HLA-B51 A, P, G F, Y, I, V HLA-B52 Q F, Y I, VHLA-B53 P W, F, L HLA-B54 P HLA-B55 P A, V HLA-B56 P A, V HLA-B57 A, T,S F, W, Y HLA-B58 A, T, S F, W, Y HLA-B67 P L HLA-B73 R P HLA-Cw1 A, L LHLA-Cw2 A, L F, Y HLA-Cw3 A, L L, M HLA-Cw4 Y, P, F L, M, F, Y HLA-Cw6L, I, V, Y HLA-Cw6 Y L, Y, F HLA-Cw8 Y L, I, HLA-Cw16 A, L L, V * In oneembodiment there is no specific anchor residue for this position,however in a preferred embodiment the anchor residue is R or A.

Thus, as an example, nonapeptides potentially having the ability to bindto HLA-A3 would have one of the following sequences:Xaa-L-Y-Xaa-Xaa-Xaa-Xaa-Xaa-K, Xaa-L-Y-Xaa-Xaa-Xaa-Xaa-Xaa-Y;Xaa-L-Y-Xaa-Xaa-Xaa-Xaa-Xaa-F or Xaa-V-Y-Xaa-Xaa-Xaa-Xaa-Xaa-K (Xaaindicating any amino acid residue). In a similar manner, sequencespotentially having the ability to bind to any other HLA molecule can bedesigned. It will be appreciated that the person of ordinary skill inthe art will be able to identify further “anchor residue motifs” for agiven HLA molecule.

The peptide of the invention may have a sequence which is a nativesequence of the CCL22 from which is derived. However, peptides having ahigher affinity to any given HLA molecule may be derived from such anative sequence by modifying the sequence by substituting, deleting oradding at least one amino acid residue, e.g. on the basis of theprocedure described above, whereby anchor residue motifs in respect ofthe given HLA molecule are identified.

Thus, in useful embodiments, the polypeptides of the invention includepeptides, the sequences of which comprise, for each of the specific HLAalleles listed in the table, any of the amino acid residues as indicatedin the table.

Thus, the peptides of the invention may be any of the above-mentionedpeptides comprising consecutive sequences from CCL22, wherein in therange of 1 to 10, preferably in the range of 1 to 5, more preferably inthe range of 1 to 3, even more preferably in the range of 1 to 2, yetmore preferably 1 amino acid has been exchanged for another amino acid,preferably in a manner so that the peptide comprises one or more,preferably all anchor residues of a given HLA-A specific peptide asindicated in the table above.

Examples of preferable HLA species, to which preferred peptides of thepresent invention are restricted include: a MHC Class I HLA speciesselected from the group consisting of HLA-A1, HLA-A2, HLA-A3, HLA-A11and HLA-A24, more preferably the peptide is restricted by HLA-A3 orHLA-A2. Alternatively a preferred HLA species includes MHC Class I HLA-Bspecies selected from the group consisting of HLA-B7, HLA-B35, HLA-B44,HLA-B8, HLA-B15, HLA-B27 and HLA-B51.

An approach to identifying polypeptides of the invention includes thefollowing steps: selecting a particular HLA molecule, e.g. one occurringat a high rate in a given population, carrying out an alignment analysisas described above to identify “anchor residue motifs” in the CCL22protein, isolating or constructing peptides of a suitable size thatcomprise one or more of the identified anchor residues and testing theresulting peptides for the capability of the peptides to elicitINF-γ-producing cells in a PBL population of a cancer patient at afrequency of at least 1 per 10⁴ PBLs as determined by an ELISPOT assayas described in Example 1. For example, the capability of the peptidesto elicit INF-γ-producing cells in a PBMC population of a cancer patienthas frequency of at least 1 per 10⁴ PBMCs.

In one aspect of the present invention, CCL22-derived peptides longerthan 8 to 10 amino acid residues are provided. Polypeptides longer than8 to 10 amino acids are processed by the proteasome to a shorter lengthfor binding to HLA molecules. Thus, when administering a polypeptidelonger than 8 to 10 amino acid residues long, the “long”polypeptide/protein/protein fragment/variant of CCL22 may be processedin vivo into a series of smaller peptides in the cytosol by theproteasome. An advantage of using a longer polypeptide that may beprocessed by the proteasome into a variety of different shorter peptidesis that more HLA classes may be targeted with one peptide than one 8 to10 amino acid peptide that is restricted to a particular HLA class.

Surprisingly, some of the peptides of the present invention bind to MHCmolecules with an affinity sufficiently high to render substitutionsunnecessary and are ready for use as antigens as they are presentedhere. Preferably, the vaccine composition of the present inventioncomprises one or more of the following: CCL22 protein (SEQ ID NO: 12 orSEQ ID NO: 15), polypeptide fragments here from, likewise variants,functional homologues of full length and partial length CCL22,contiguous peptides of CCL22 and functional homologues of these. Morepreferably, the vaccine composition comprises any of the sequenceslisted in Table 1. Very preferably, the vaccine composition comprisesthe peptides SEQ ID NO: 1 (CCL22₁₋₂₂), SEQ ID NO: 3 (CCL22₃₋₁₁) SEQ IDNO: 4 (CCL22₃₋₁₂), SEQ ID NO: 11 (CCL22₁₋₂₄), SEQ ID NO: 13 (mCCL22₁₋₂₂)or SEQ ID NO: 14 (mCCL22₁₋₂₄).

A significant feature of the peptide of the invention is its capabilityto recognize or elicit INF-γ-producing responder T cells, i.e. cytotoxicT cells (CTLs) that specifically recognize the particular peptide in aPBL population, on an APC or tumor/neoplastic cells of an individualsuffering from a cancer and/or an infection (target cells). Thisactivity is readily determined by subjecting PBLs, PBMCs, APCs or tumorcells from an individual to an ELISPOT assay. Prior to the assay, it maybe advantageous to stimulate the cells to be assayed by contacting thecells with the peptide to be tested. Preferably, the peptide is capableof eliciting or recognizing INF-γ-producing T cells at a frequency of atleast 1 per 10⁴ PBLs such as at a frequency of at least 1 per 10⁴ PBMCsas determined by an ELISPOT assay as used herein. More preferably thefrequency is at least 5 per 10⁴ PBLs, most preferably at least 10 per10⁴ PBLs, such as at least 50 or 100 per 10⁴ PBLs. For example, thefrequency is at least 5 per 10⁴ PBMCs, most preferably at least 10 per10⁴ PBMCs, such as at least 50 or 100 per 10⁴ PBMCs.

The ELISPOT assay represents a strong tool to monitor CCL22 peptidespecific T-cell responses. A major implication of the findings herein isthat the peptides of the invention are expressed and complexed with HLAmolecules on cancer cells and/or CCL22 expressing APCs. This rendersthese cancer cells susceptible to destruction by CTLs and emphasizes theusefulness of CCL22 immunization to fight cancer and infections. Thepresence of spontaneous CTL-responses in PBLs from melanoma patients toHLA-restricted CCL22 derived peptide epitopes shows theimmunotherapeutic potential of CCL22 immunogenic peptides.

In an embodiment of the present invention the peptide of the inventionis capable of eliciting INF-γ-producing cells in a PBL population of anindividual suffering from an clinical condition where CCL22 of SEQ IDNO: 12 or a functional homologue thereof having at least 70% identity toSEQ ID NO: 12 is expressed. The clinical condition is preferably acancer and/or an infection and most preferably a cancer.

Individual

The individual to be treated with the vaccine composition of the presentinvention is an individual suffering from a clinical condition. Theindividual is preferably of a mammalian species and most preferably ahuman being. The individual may be of any age, young or old, and may beeither male or female. The clinical condition from which the individualsuffers may be a neoplastic disease such as a cancer, or an infectionsuch as a microbial or viral infection e.g. HIV.

An embodiment of the present invention provides a vaccine for thetreatment, reduction of risk of, stabilization of or prevention of acancer. In another embodiment the present invention provides a vaccinefor the treatment, reduction of risk of, stabilization of or preventionof a disease stemming from an infection, such as a microbial or viralinfection.

Cancer

The vaccine composition of the present invention may be used to prevent,reduce the risk of or treat a clinical condition. Preferably, theclinical condition is associated with or characterized by the expressionof CCL22. CCL22 may be CCL22 as identified in SEQ ID NO: 12 or may be ahomolog sharing at least 70% identity therewith in their wild typeforms, but need not be functional. It is understood hereby that theexpression level of CCL22 (the expression being expression of e.g.hnRNA, mRNA, precursor protein, fully processed protein) is the same orhigher than in an individual not suffering from a clinical condition.

In one embodiment of the invention the clinical condition is aproliferative disorder, such as a preneoplastic or neoplastic disorder.In a preferred embodiment of the invention, the clinical condition iscancer. Cancer (malignant neoplasm) is a class of diseases in which agroup of cells display the traits of uncontrolled growth (growth anddivision beyond the normal limits), invasion (intrusion on anddestruction of adjacent tissues), and sometimes metastasis (spread toother locations in the body via lymph or blood). These three malignantproperties of cancers differentiate them from benign tumors, which areself-limited, do not invade or metastasize. Most cancers form a tumorbut some, like leukemia, do not.

A non-limiting group of cancers given as examples of cancers that may betreated, managed and/or prevented by administration of the vaccine ofthe present invention include: colon carcinoma, breast cancer,pancreatic cancer, ovarian cancer, prostate cancer, fibrosarcoma,myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma,angiosarcoma, endotheliosarcoma, lymphangeosarcoma, lymphangeoendotheliasarcoma, synovioma, mesothelioma, Ewing's sarcoma, leiomyosarcoma,rhabdomyosarcoma, squamous cell carcinoma, basal cell carcinoma,adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma,papillary carcinoma, papillary adenocarcinomas, cystandeocarcinoma,medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma,hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonalcarcinoma, Wilms' tumor, cervical cancer, testicular tumor, lungcarcinoma, small cell lung carcinoma, bladder carcinoma, epithelialcarcinoma, glioblastomas, neuronomas, craniopharingiomas, schwannomas,glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma,pinealoma, hemangioblastoma, acoustic neuroama, oligodendroglioma,meningioma, melanoma, neuroblastoma, retinoblastoma, leukemias andlymphomas, acute lymphocytic leukemia and acute myelocytic polycythemiavera, multiple myeloma, Waldenstrom's macroglobulinemia, and heavy chaindisease, acute nonlymphocytic leukemias, chronic lymphocytic leukemia,chronic myelogenous leukemia, Hodgkin's Disease, non-Hodgkin'slymphomas, rectum cancer, urinary cancers, uterine cancers, oralcancers, skin cancers, stomach cancer, brain tumors, liver cancer,laryngeal cancer, esophageal cancer, mammary tumors, childhood-nullacute lymphoid leukemia (ALL), thymic ALL, B-cell ALL, acute myeloidleukemia, myelomonocytoid leukemia, acute megakaryocytoid leukemia,Burkitt's lymphoma, acute myeloid leukemia, chronic myeloid leukemia,and T cell leukemia, small and large non-small cell lung carcinoma,acute granulocytic leukemia, germ cell tumors, endometrial cancer,gastric cancer, cancer of the head and neck, chronic lymphoid leukemia,hairy cell leukemia and thyroid cancer.

In a preferred embodiment the vaccine composition according to theinvention is capable of eliciting a clinical response in subject,wherein the clinical response may be characterized by a stable disease,in a preferred embodiment the clinical response may be characterized bya partial response or preferably the clinical response may becharacterized by complete remission of a cancer. Preferably, the canceris selected from the group of; melanoma, breast cancer, ovarian cancer,lung cancer, pancreatic cancer, hematologic cancers (such as leukemias),colon and renal cell cancers.

In one aspect of the invention the vaccine composition is capable ofeliciting a clinical response in an individual. In one embodiment theclinical response may be characterized by a stable disease (no furtherworsening or progression), in a preferred embodiment the clinicalresponse may be characterized by a partial response or preferably theclinical response may be characterized by complete remission of a canceror infections. The clinical response may be determined as describedherein below.

In another aspect of the invention the vaccine composition is capable ofeliciting a clinical response in subject, wherein the clinical responseis characterized by a decrease in the sum of the longest diameter of thelargest target lesion. The decrease may be determined as describedherein below.

All measurable lesions up to a maximum of five lesions per organ and 10lesions in total, representative of all involved organs should beidentified as target lesions and recorded and measured at baseline.

-   -   Target lesions should be selected on the basis of their size        (lesions with the longest diameter) and their suitability for        accurate repeated measurements (either by imaging techniques or        clinically).    -   A sum of the longest diameter (LD) for all target lesions will        be calculated and reported as the baseline sum LD. The baseline        sum LD will be used as reference by which to characterize the        objective tumor.    -   All other lesions (or sites of disease) should be identified as        non-target lesions and should also be recorded at baseline.        Measurements of these lesions are not required, but the presence        or absence of each should be noted throughout follow-up.        Evaluation of Target Lesions    -   Complete Response (CR): Disappearance of all target lesions    -   Partial Response (PR): At least a 30% decrease in the sum of the        LD of target lesions, taking as reference the baseline sum LD    -   Progressive Disease (PD): At least a 20% increase in the sum of        the LD of target lesions, taking as reference the smallest sum        LD recorded since the treatment started or the appearance of one        or more new lesions    -   Stable Disease (SD): Neither sufficient shrinkage to qualify for        PR nor sufficient increase to qualify for PD, taking as        reference the smallest sum LD since the treatment started        Evaluation of Non-Target Lesions    -   Complete Response (CR): Disappearance of all non-target lesions        and normalization of tumor marker level    -   Incomplete Response/Stable Disease (SD): Persistence of one or        more non-target lesion(s) or/and maintenance of tumor marker        level above the normal limits    -   Progressive Disease (PD): Appearance of one or more new lesions        and/or unequivocal progression of existing non-target lesions

In an embodiment of the present invention the vaccine compositioncomprising any of the herein mentioned proteins and/or polypeptides iscapable of eliciting a clinical response in a subject, wherein theclinical response is characterized by a decrease in the sum of thelongest diameter of the largest target lesion

It is contemplated that the vaccine composition of the invention iscapable of eliciting an immune response against a cancer expressingCCL22 of SEQ ID NO: 12 or a functional homologue thereof having at least70% identity to SEQ ID NO: 12, when administered to an individualsuffering from a cancer expressing CCL22. The vaccine composition of theinvention is capable of eliciting the production in a vaccinatedindividual of effector T-cells having a cytotoxic effect against thecancer cells, CCL22 expressing APCs and/or inducing infiltration ofantigen specific T-cells in tumor stroma in a subject.

In addition to their capacity to elicit immune responses in PBLpopulations it is also contemplated that the peptides of the inventionare capable of eliciting cytolytic immune responses in situ, i.e. insolid tumor tissues. This may for example be demonstrated by providingHLA-peptide complexes, e.g. being multimerized and being provided with adetectable label, and using such complexes for immunohistochemistrystainings to detect in a tumor tissue CTLs that are reactive with theepitope peptide of the invention. Accordingly, a further significantfeature of the peptide of the invention is that it is capable of in situdetection in a tumor tissue of CTLs that are reactive with the epitopepeptide.

It is also contemplated that the peptides of the invention, in additionto their capacity to bind to HLA molecules resulting in the presentationof complexes of HLA and peptides on cell surfaces, which complexes inturn act as epitopes or targets for cytolytic T cells, may elicit othertypes of immune responses, such as B-cell responses resulting in theproduction of antibodies against the complexes and/or a Delayed TypeHypersensitivity (DTH) reaction. The latter type of immune response isdefined as a redness and palpable induration at the site of injection ofthe peptide of the invention.

It is an object of the presenting invention to provide a vaccinecomposition comprising C-C motif chemokine 22 of SEQ ID NO: 12 or SEQ IDNO: 15 or a functional homologue thereof having at least 70% identity toSEQ ID NO: 12 or SEQ ID NO: 15 or an immunogenically active peptidefragment comprising a consecutive sequence of said CCL22 or saidfunctional homologue thereof or a nucleic acid encoding said CCL22 orsaid peptide fragment; and an adjuvant, for the prevention of, reductionof risk from or treatment of cancer.

Cancer Combination Treatment

In some cases it will be appropriate to combine the treatment method ofthe invention with a further cancer treatment such as chemotherapy,radiotherapy, treatment with immunostimulating substances, gene therapy,treatment with antibodies and treatment using dendritic cells.

Since elevated expression of CCL22 in tumor cells leads to inhibition ofthe immune system, the combination of a CCL22-based immunotherapy asdisclosed by the present invention with cytotoxic chemotherapy and oranother anti-cancer immunotherapeutic treatment is an effective approachto treat cancer. These remedies are also referred to herein as “secondactive ingredients”.

Examples of chemotherapeutic agents that are of relevance in regards toco-administration (sequentially or simultaneously) with the vaccinecomposition of the present invention include, but are not limited to:all-trans retinoic acid, Actimide, Azacitidine, Azathioprine, Bleomycin,Carboplatin, Capecitabine, Cisplatin, Chlorambucil, Cyclophosphamide,Cytarabine, Daunorubicin, Docetaxel, Doxifluridine, Doxorubicin,Epirubicin, Etoposide, Fludarabine, Fluorouracil, Gemcitabine,Hydroxyurea, Idarubicin, Irinotecan, Lenalidomide, Leucovorin,Mechlorethamine, Melphalan, Mercaptopurine, Methotrexate, Mitoxantrone,Oxaliplatin, Paclitaxel, Pemetrexed, Revlimid, Temozolomide, Teniposide,Thioguanine, Valrubicin, Vinblastine, Vincristine, Vindesine andVinorelbine. In one embodiment, a chemotherapeutic agent for use in thecombination of the present agent may, itself, be a combination ofdifferent chemotherapeutic agents. Suitable combinations include FOLFOXand IFL. FOLFOX is a combination which includes 5-fluorouracil (5-FU),leucovorin, and oxaliplatin. IFL treatment includes irinotecan, 5-FU,and leucovorin.

Another second active ingredient may be a kinase inhibitor, forseparate, simultaneous or combined use in the treatment of tumors.Suitable kinase inhibitors include those which have been shown topossess anti-tumor activity (such as gefitinib (Iressa) and erlotinib(Tarceva) and these could be used in combination with the peptides. Thereceptor tyrosine kinase inhibitors, such as Sunitinib malate andSorafenib which have been shown to be effective in the treatment ofrenal cell carcinoma are also suitable to be used as second activeingredients.

Further examples of second active ingredients are immunostimulatingsubstances e.g. cytokines and antibodies. Such as cytokines may beselected from the group consisting of, but not limited to: GM-CSF, typeI IFN, interleukin 21, interleukin 2, interleukin 12 and interleukin 15.The antibody is preferably an immunostimulating antibody such asanti-CD40 or anti-CTLA-4 antibodies. The immunostimulatory substance mayalso be a substance capable of depletion of immune inhibitory cells(e.g. regulatory T-cells) or factors, said substance may for example beE3 ubiquitin ligases. E3 ubiquitin ligases (the HECT, RING and U-boxproteins) have emerged as key molecular regulators of immune cellfunction, and each may be involved in the regulation of immune responsesduring infection by targeting specific inhibitory molecules forproteolytic destruction.

Several HECT and RING E3 proteins have now also been linked to theinduction and maintenance of immune self-tolerance: c-Cbl, Cbl-b, GRAIL,Itch and Nedd4 each negatively regulate T cell growth factor productionand proliferation.

In an embodiment, the vaccine composition of the present invention,comprising a CCL22 derived polypeptide, is administered in combinationwith a second active ingredient, such as an immunostimulatory substance.The immunostimulatory substance is preferably an interleukin such asIL-21 or IL-2 or a chemotherapeutic agent.

The vaccine compositions of the invention may also comprise one or moreadditional antigens in addition to CCL22. Said antigens, may for examplebe immunogenically active peptides derived from cancer associatedproteins.

Thus, the vaccine compositions of the invention may in addition to CCL22and/or immunogenically active peptide fragments thereof also compriseone or more of the following:

-   -   1) Indoleamine-2,3-dioxygenase (IDO)    -   2) An immunogenically active peptide fragment of IDO    -   3) A functional homologue of 1) or 2)    -   4) A polypeptide comprising 1), 2) or 3)    -   5) A nucleic acid encoding any of 1), 2), 3) or 4).

Said IDO may in particular be IDO of SEQ ID NO: 1 of WO 2009/143843, IDOof SEQ ID NO: 13 of WO 2009/143843, IDO of SEQ ID NO: 14 of WO2009/143843, IDO of SEQ ID NO: 15 of WO 2009/143843 or IDO of SEQ ID NO:16 of WO 2009/143843. Useful immunogenically active peptide fragments ofIDO, which can be contained in the vaccine compositions of the presentinvention are described in WO 2009/143843.

The vaccine compositions of the invention may in addition to CCL22and/or immunogenically active peptide fragments thereof also compriseone or more of the following:

-   -   1) PD-L1    -   2) An immunogenically active peptide fragment of PD-L1    -   3) A functional homologue of 1) or 2)    -   4) A polypeptide comprising 1), 2) or 3)    -   5) A nucleic acid encoding any of 1), 2), 3) or 4).

Said PD-L1 may in particular be PD-L1 of SEQ ID NO: 1 of WO2013/056716.Useful immunogenically active peptide fragments of PD-L1, which can becontained in the vaccine compositions of the present invention, aredescribed in WO2013/056716.

The vaccine compositions of the invention may in addition to CCL22and/or immunogenically active peptide fragments thereof also compriseone or more of the following:

-   -   1) tryptophan 2,3-dioxygenase (TDO)    -   2) An immunogenically active peptide fragment of TDO    -   3) A functional homologue of 1) or 2)    -   4) A polypeptide comprising 1), 2) or 3)    -   5) A nucleic acid encoding any of 1), 2), 3) or 4).

Said TDO may in particular be TDO of SEQ ID NO: 1 of pending application“Vaccine compositions comprising Tryptophan 2,3-dioxygenase or fragmentsthereof” filed by the present inventors. Useful immunogenically activepeptide fragments of TDO, which can be contained in the vaccinecompositions of the present invention, are described in said pendingapplication.

Infections

In another embodiment of the invention, the vaccine compositionsdisclosed herein are for treatment or prevention of an inflammatorycondition.

The term “inflammatory condition” as used herein relates to any kind ofclinical condition giving rise to an immune response, such as aninflammation, and therefore includes infectious diseases, chronicinfections, autoimmune conditions and allergic inflammations. Thus,inflammatory conditions, such as infectious diseases, chronicinfections, autoimmune conditions and allergic inflammations are allclinical conditions of relevance for the present invention, and aredealt with in turn hereunder.

Inflammation is the complex biological response of vascular tissues toharmful stimuli, such as pathogens, damaged cells, or irritants. It is aprotective attempt by the organism to remove the injurious stimuli aswell as initiate the healing process for the tissue. Inflammation can beclassified as either acute or chronic. Acute inflammation is the initialresponse of the body to harmful stimuli and is achieved by the increasedmovement of plasma and leukocytes from the blood into the injuredtissues. A cascade of biochemical events propagates and matures theinflammatory response, involving the local vascular system, the immunesystem, and various cells within the injured tissue. Prolongedinflammation, known as chronic inflammation, leads to a progressiveshift in the type of cells which are present at the site of inflammationand is characterized by simultaneous destruction and healing of thetissue from the inflammatory process. In either case, CCL22 is expressedby cells of the immune system such as the APCs and therefore infectionsand inflammations are clinical conditions that may be treated,prevented, or from which the risk may be reduced by the administrationof the vaccine composition of the present invention. The vaccinecomposition preferably comprises CCL22 protein, protein fragments,polypeptide or peptides derived there from or functional homologues ofany of these.

Examples of disorders associated with inflammation which are ofrelevance to the presenting invention include, but are not limited to:Allergic inflammations, Asthma, Autoimmune diseases, Chronicinflammations, Chronic prostatitis, Glomerulonephritis,Hypersensitivities, Infectious diseases, Inflammatory bowel diseases,Pelvic inflammatory disease, Reperfusion injury, Rheumatoid arthritis,Transplant rejection, and Vasculitis.

Chronic Inflammations

Chronic inflammation is especially of relevance in regard to the presentinvention. A chronic inflammation is a pathological conditioncharacterized by concurrent active inflammation, tissue destruction, andattempts at repair. Chronically inflamed tissue is characterized by theinfiltration of mononuclear immune cells (monocytes, macrophages,lymphocytes, and plasma cells), tissue destruction, and attempts athealing, which include angiogenesis and fibrosis.

In acute inflammation, removal of the stimulus halts the recruitment ofmonocytes (which become macrophages under appropriate activation) intothe inflamed tissue, and existing macrophages exit the tissue vialymphatics. However in chronically inflamed tissue the stimulus ispersistent, and therefore recruitment of monocytes is maintained,existing macrophages are tethered in place, and proliferation ofmacrophages is stimulated (especially in atheromatous plaques).

It is an object of the presenting invention to provide a vaccinecomposition comprising CCL22 of SEQ ID NO: 12 or SEQ ID NO: 15 or afunctional homologue thereof having at least 70% identity to SEQ ID NO:12 or SEQ ID NO: 15 or an immunogenically active peptide fragmentcomprising a consecutive sequence of said CCL22 or said functionalhomologue thereof or a nucleic acid encoding said CCL22 or said peptidefragment; and an adjuvant, for the prevention of, reduction of risk fromor treatment of chronic inflammations.

Infectious Diseases

The vaccine composition of the present invention may be used to prevent,reduce the risk from or treat a clinical condition. In a preferredembodiment of the invention, the clinical condition is an infectiousdisease. The infectious disease may be promoted by any infectious agentsuch as bacteria, virus, parasites and or fungi that are capable ofinducing an increased expression of CCL22 in the individual sufferingfrom the infectious disease; preferably, the infectious disease is or isat risk of becoming a chronic disease. As described in the background ofinvention, the increased expression of CCL22 has an immediate effect onthe microbial agents in the vicinity of the CCL22 expressing organism bydepriving it of tryptophan. However, this approach backfires, as theincreased CCL22 expression induces inhibits the activity of Treg cells,if the CCL22 expressing cell is an APC. Therefore it is an aspect of thepresent invention to provide a vaccine composition comprising CCL22protein, protein fragments, peptides and or variant of any of these forthe treatment, amelioration of (lessening of severity) stabilizationand/or prevention of a disease caused by an infectious agent.

An infectious diseases may be caused by a virus, and viral diseasesagainst which the vaccine composition of the present invention may beadministered in the treatment of include, but are not limited to thefollowing viral diseases: HIV, AIDS, AIDS Related Complex, Chickenpox(Varicella), Common cold, Cytomegalovirus Infection, Colorado tickfever, Dengue fever, Ebola hemorrhagic fever, Hand, foot and mouthdisease, Hepatitis, Herpes simplex, Herpes zoster, HPV (Humanpapillomavirus), Influenza (Flu), Lassa fever, Measles, Marburghemorrhagic fever, Infectious mononucleosis, Mumps, Norovirus,Poliomyelitis, Progressive multifocal leukencephalopathy, Rabies,Rubella, SARS, Smallpox (Variola), Viral encephalitis, Viralgastroenteritis, Viral meningitis, Viral pneumonia, West Nile disease,and Yellow fever. Preferably, the vaccine composition is administered toindividuals suffering from HIV/AIDS and viral infections that may causecancer. The main viruses associated with human cancers are humanpapillomavirus, hepatitis B and hepatitis C virus, Epstein-Barr virus,and human T-lymphotropic virus; thus it is an object of the presentinvention to be administered as the treatment of or as part of thetreatment of these viral infections.

Examples of bacterial infections of relevance for the present inventioninclude, but are not limited to: Anthrax, Bacterial Meningitis,Botulism, Brucellosis, Campylobacteriosis, Cat Scratch Disease, Cholera,Diphtheria, Epidemic Typhus, Gonorrhea, Impetigo, Legionellosis, Leprosy(Hansen's Disease), Leptospirosis, Listeriosis, Lyme disease,Melioidosis, Rheumatic Fever, MRSA infection, Nocardiosis, Pertussis(Whooping Cough), Plague, Pneumococcal pneumonia, Psittacosis, Q fever,Rocky Mountain Spotted Fever (RMSF), Salmonellosis, Scarlet Fever,Shigellosis, Syphilis, Tetanus, Trachoma, Tuberculosis, Tularemia,Typhoid Fever, Typhus, and Urinary Tract Infections. It is an object ofthe present invention to provide a vaccine for the treatment and/orprevention and/or reduction of risk from a bacterial infection.

It is a further aspect of the present invention to provide a vaccinecomposition for the treatment and/or prevention and/or reduction of riskfrom: Parasitic infectious diseases such as, but not limited to: Africantrypanosomiasis, Amebiasis, Ascariasis, Babesiosis, Chagas Disease,Clonorchiasis, Cryptosporidiosis, Cysticercosis, Diphyllobothriasis,Dracunculiasis, Echinococcosis, Enterobiasis, Fascioliasis,Fasciolopsiasis, Filariasis, Free-living amebic infection, Giardiasis,Gnathostomiasis, Hymenolepiasis, Isosporiasis, Kala-azar, Leishmaniasis,Malaria, Metagonimiasis, Myiasis, Onchocerciasis, Pediculosis, PinwormInfection, Scabies, Schistosomiasis, Taeniasis, Toxocariasis,Toxoplasmosis, Trichinellosis, Trichinosis, Trichuriasis,Trichomoniasis, and Trypanosomiasis; Fungal infectious diseases such asbut not limited to: Aspergillosis, Blastomycosis, Candidiasis,Coccidioidomycosis, Cryptococcosis, Histoplasmosis, Tinea pedis; Prioninfectious diseases such as but not limited to: transmissible spongiformencephalopathy, Bovine spongiform encephalopathy, Creutzfeldt-Jakobdisease, Kuru-Fatal Familial Insomnia, and Alpers Syndrome; thus it isan object of the present invention to be administered as the treatmentof or as part of the treatment of these parasitic, fungal or prioncaused infections.

Infectious Disease Combination Treatment

It is further provided for that a treatment of any infectious disease bythe administration of the vaccine composition according to the presentinvention may be given in conjunction with a further (second) activeingredient or in combination with a further treatment such as antibiotictreatment, chemotherapy, treatment with immunostimulating substances,treatment using dendritic cells, antiviral agents anti parasitic agentsand so forth.

Examples of a second active ingredient that may be used in the treatmentof an infectious disease in combination with the vaccine of the presentinvention include, and are not limited to antibiotics. The termantibiotics herein refers to substances with anti-bacterial,anti-fungal, anti-viral and/or anti-parasitical activity; examples ofrelevance to the present invention include, but are not limited to:Amikacin, Gentamycin, Kanamycin, Neomycin, Netilmicin, Paromomycin,Streptomycin, Tobramycin, Ertapenem, Imipenem, Meropenem,Chloramphenicol, Fluoroquinolones, Ciprofloxacin, Gatifloxacin,Gemifloxacin, Grepafloxacin, Levofloxacin, Lomefloxacin, Moxifloxacin,Norfloxacin, Ofloxacin, Sparfloxacin, Trovafloxacin, Glycopeptides,Vancomycin, Lincosamides, Clindamycin, Macrolides/Ketolides,Azithromycin, Clarithromycin, Dirithromycin, Erythromycin, Cefadroxil,Cefazolin, Cephalexin, Cephalothin, Cephapirin, Cephradine, Cefaclor,Cefamandole, Cefonicid, Cefotetan, Cefoxitin, Cefprozil, Cefuroxime,Loracarbef, Cefdinir, Cefditoren, Cefixime, Cefoperazone, Cefotaxime,Cefpodoxime, Ceftazidime, Ceftibuten, Ceftizoxime, Ceftriaxone,Cefepime, Monobactams, Aztreonam, Nitroimidazoles, Metronidazole,Oxazolidinones, Linezolid, Penicillins, Amoxicillin,Amoxicillin/Clavulanate, Ampicillin, Sulbactam, Bacampicillin,Carbenicillin, Cloxacillin, Dicloxacillin, Methicillin, Mezlocillin,Nafcillin, Oxacillin, Penicillin G, Penicillin V, Piperacillin,Piperacillin/Tazobactam, Ticarcillin, Ticarcillin/Clavulanate,Streptogramins, Quinupristin, Dalfopristin,Sulfonamide/Sulfamethoxazole, Trimethoprim, Tetracyclines,Demeclocycline, Doxycycline, Minocycline, Tetracycline, Azoleantifungals Clotrimazole Fluconazole, Itraconazole, Ketoconazole,Miconazole, Voriconazole, Amphotericin B, Nystatin, Echinocandin,Caspofungin, Micafungin, Ciclopirox, Flucytosine, Griseofulvin, andTerbinafine. Of further relevance are antivirals such as Vidarabine,Acyclovir, Gancyclovir and Valcyte (valganciclovir), Nucleoside-analogreverse transcriptase inhibitors (NRTI): AZT (Zidovudine), ddl(Didanosine), ddC (Zalcitabine), d4T (Stavudine), 3TC (Lamivudine),Non-nucleoside reverse transcriptase inhibitors (NNRTI): Nevirapine,Delavirdine, Protease Inhibitors: Saquinavir, Ritonavir, Indinavir,Nelfinavir, Ribavirin, Amantadine/Rimantadine, Relenza and Tamiflu,Pleconaril, Interferons

In an embodiment, the present invention regards a vaccine compositioncomprising CCL22 derived proteins, polypeptides and/or functionalhomologs of these for the treatment of an infectious disease incombination with at least one antibiotic. Preferably, the vaccinecomposition of the present invention is used for the treatment ofchronic infections e.g. HIV and therefore is used in combination withany of the above listed antibiotics such as anti-viral agents.

Autoimmune Diseases

Autoimmune diseases arise when an organism fails to recognize its ownconstituent parts (down to the sub-molecular levels) as self, whichresults in an immune response against its own cells and tissues. Anydisease that results from such an aberrant immune response is termed anautoimmune disease and is of relevance to the present invention.Examples hereof include but are not limited to: Coeliac disease,diabetes mellitus type 1 (IDDM), systemic lupus erythematosus (SLE),Sjögren's syndrome, multiple sclerosis (MS), Hashimoto's thyroiditis,Graves' disease, idiopathic thrombocytopenic purpura, and rheumatoidarthritis (RA).

It is an object of the present invention to provide a vaccinecomposition comprising CCL22 of SEQ ID NO: 12 or SEQ ID NO: 15 or afunctional homologue thereof having at least 70% identity to SEQ ID NO:12 or SEQ ID NO: 15 or an immunogenically active peptide fragmentcomprising a consecutive sequence of said CCL22 or said functionalhomologue thereof or a nucleic acid encoding said CCL22 or said peptidefragment; and an adjuvant, for the prevention of, reduction of risk fromor treatment of autoimmune diseases.

Autoimmune Disease Combination Treatment

Current treatments for autoimmune disease are usually immunosuppressive,anti-inflammatory, or palliative. Dietary manipulation limits theseverity of celiac disease. Steroidal or NSAID treatment limitsinflammatory symptoms of many diseases. Intravenous preparations ofimmune globulin (IVIG) are used for Chronic Inflammatory DemyelinatingPolyneuropathy (CIDP) and Guillain-Barré syndrome (GBS). More specificimmunomodulatory therapies, such as the TNFα antagonist Etanercept, havebeen shown to be useful in treating RA. These immunotherapies may beassociated with increased risk of adverse effects, such assusceptibility to infection.

Helminthic therapy has developed based on these observations andinvolves inoculation of the individual with specific parasiticintestinal nematodes (helminths). There are currently twoclosely-related treatments available, inoculation with either Necatoramericanus, commonly known as hookworms, or Trichuris Suis Ova, commonlyknown as Pig Whipworm Eggs. Research is available that demonstrates thisapproach is highly effective in treating a variety of autoimmunedisorders, including Crohn's, Ulcerative Colitis, Asthma, allergies,Multiple Sclerosis, and chronic inflammatory disorders

In an embodiment, the vaccine herein disclosed is used in combinationwith a second active ingredient such as any of the above mentioned drugsand treatments against autoimmune diseases.

Allergic Inflammation

Allergy is a disorder of the immune system often also referred to asatopy. Allergic reactions occur to environmental substances known asallergens; these reactions are acquired, predictable and rapid.Strictly, allergy is one of four forms of hypersensitivity and is calledtype I (or immediate) hypersensitivity. It is characterized by excessiveactivation of certain white blood cells called mast cells and basophilsby a type of antibody, known as IgE, resulting in an extremeinflammatory response. Common allergic reactions include eczema, hives,hay fever, asthma, food allergies, and reactions to the venom ofstinging insects such as wasps and bees.

Allergic inflammation is an important pathophysiological feature ofseveral disabilities or medical conditions including allergic asthma,atopic dermatitis, allergic rhinitis and several ocular allergicdiseases.

It is an object of the present invention to provide a vaccinecomposition comprising CCL22 of SEQ ID NO: 12 or SEQ ID NO: 15 or afunctional homologue thereof having at least 70% identity to SEQ ID NO:12 or SEQ ID NO: 15 or an immunogenically active peptide fragmentcomprising a consecutive sequence of said CCL22 or said functionalhomologue thereof or a nucleic acid encoding said CCL22 or said peptidefragment; and an adjuvant, for the prevention of, reduction of risk fromor treatment of allergic inflammation.

Allergic Inflammation Combination Treatment

Two types of treatments are available for the treatment of allergicinflammations, pharmacotherapy and immunotherapy: pharmacotherapy andimmunotherapy.

Pharmacotherapy is the use of antagonistic drugs to block the action ofallergic mediators, or to prevent activation of cells and degranulationprocesses. These include antihistamines, cortisone, dexamethasone,hydrocortisone, epinephrine (adrenaline), theophylline, cromolyn sodiumand anti-leukotrienes, such as Montelukast (Singulair) or Zafirlukast(Accolate); anti-cholinergics, decongestants, mast cell stabilizers, andother compounds thought to impair eosinophil chemotaxis, are alsocommonly used.

Immunotherapy is the desensitization or hyposensitization treatment inwhich the individual is gradually vaccinated with progressively largerdoses of the allergen in question. A second form of immunotherapyinvolves the intravenous injection of monoclonal anti-IgE antibodies. Athird type, Sublingual immunotherapy, is an orally-administered therapywhich takes advantage of oral immune tolerance to non-pathogenicantigens such as foods and resident bacteria.

In an embodiment, the vaccine herein disclosed is used in combinationwith a second active ingredient such as any of the above mentioned drugsand treatments against allergic inflammations.

Pharmaceutical Compositions

The present invention regards pharmaceutical compositions capable oftreating, reducing the risk of and/or preventing a clinical disorderassociated with CCL22 expression in an individual. Said pharmaceuticalcomposition may in particular be a vaccine composition. The vaccinecompositions of the present invention may be “traditional” vaccinecompositions comprising antigens such as proteins polypeptides and/ornucleic acid molecules. They may also be in the form of compositionscomprising cells, such as modified cells originating from the individualand later processed, or to compositions comprising complex moleculessuch as antibodies or TCRs.

Generally, a vaccine is a substance or composition capable of inducingan immune response in an individual. The composition may comprise one ormore of the following: an “active component” such as an antigen(s) (e.g.protein, polypeptides, peptides, nucleic acids and the like), nucleicacid constructs comprising one or more antigens amongst other elements,cells, (e.g. loaded APC, T cells for adoptive transder aso.), complexmolecules (Antibodies, TCRs and MHC complexes and more), carriers,adjuvants and pharmaceutical carriers. In the following, the variouscomponents of a vaccine composition according to the present inventionare disclosed in more detail.

The vaccine composition of the invention is capable of eliciting animmune response against a cancer, DC or APC expressing CCL22 of SEQ IDNO: 12 or SEQ ID NO: 15 or a functional homologue thereof having atleast 70% identity to SEQ ID NO: 12 or SEQ ID NO: 15, when administeredto an individual suffering from a cancer and/or infection (leading tothe expression of CCL22). In a preferred embodiment the clinicalcondition is a cancer. The vaccine composition of the invention iscapable of eliciting the production in a vaccinated individual ofeffector T-cells having a cytotoxic effect against cancer cells, APCsand DCs expressing CCL22 and/or inducing infiltration of antigenspecific T-cells in tumor stroma in a subject.

Antigens and Other Active Components

Protein/Polypeptide Based Vaccine Compositions

The peptides of the present invention are ready for use as antigens asthey are presented here. Preferably, the vaccine composition of thepresent invention comprises one or more of the following:

-   -   1) C-C motif chemokine 22 (CCL22), which may be any of the        CCL22s described herein in the section “C-C motif chemokine 22”,        in particular human or murine CCL22;    -   2) An immunogenically active peptide fragment of CCL22        comprising a consecutive sequence of amino acids of CCL22, which        may be any of the peptides described herein below in the section        “Immunogenically active peptide fragment of CCL22”.    -   3) An immunogenically active peptide fragments of CCL22, which        is an MHC Class I-restricted peptide fragment or MHC Class        II-restricted peptide fragment, such as any of the an MHC Class        I-restricted peptide fragments or MHC Class II-restricted        peptide fragments described in the section “MHC”;    -   4) A functional homologue of the polypeptides under 1), 2) and        3);    -   5) A polypeptide comprising any of polypeptides under 1), 2), 3)        and 4), which may be any of the polypeptides described herein        below in the section “Polypeptides comprising CCL22 or a        fragment thereof”;    -   6) A nucleic acid encoding any of the polypeptides under 1),        2), 3) and 4).

The choice of antigen in the vaccine composition of the invention willdepend on parameters determinable by the person of skill in the art. Asit has been mentioned, each of the different peptides of the inventionis presented on the cell surfaces by a particular HLA molecule. As such,if a subject to be treated is typed with respect to HLA phenotype, apeptide/peptides are selected that is/are known to bind to thatparticular HLA molecule. Alternatively, the antigen of interest isselected based on the prevalence of the various HLA phenotypes in agiven population. As an example, HLA-A2 is the most prevalent phenotypein the Caucasian population, and therefore, a composition containing apeptide binding to HLA-A2 will be active in a large proportion of thatpopulation. Furthermore, the antigens/peptides of the present inventionmay be modified according to the anchor residue motifs presented inTable 2, to enhance binding to particular HLA molecules.

The composition of the invention may also contain a combination of twoor more immunogenically active peptide fragments of CCL22 e.g. any ofthe peptides described in the sections “Immunogenically active peptidefragments of CCL22”, “Polypeptides comprising CCL22 or a fragmentthereof” and “MHC”. Said immunogenically active peptide fragments ofCCL22 may each interact specifically with a different HLA molecule so asto cover a larger proportion of the target population. Thus, asexamples, the pharmaceutical composition may contain a combination of apeptide restricted by a HLA-A molecule and a peptide restricted by aHLA-B molecule, e.g. including those HLA-A and HLA-B molecules thatcorrespond to the prevalence of HLA phenotypes in the target population,such as e.g. HLA-A2 and HLA-B35. Additionally, the composition maycomprise a peptide restricted by an HLA-C molecule.

In the case of peptide-based vaccines, epitopes can be administered inan ‘MHC-ready’ form, which enables presentation through exogenousloading independently of antigen uptake and processing by hostantigen-presenting cells. The peptides of the present invention compriseboth peptides in a short ‘MHC-ready’ form and in a longer form requiringprocessing by the proteasome thus providing a more complex vaccinecomposition that can target multiple tumor antigens. The more differentHLA groups are targeted by a vaccine, the higher likelihood of thevaccine functioning in diverse populations.

Multi Epitope Vaccine Composition

The invention also relates to highly immunogenic multi-epitope vaccines.Preferably, such vaccines should be designed so as to facilitate asimultaneous delivery of the best-suited immunogenically active peptidefragments of CCL22 optionally in combination with other suitablepeptides and/or adjuvants as described hereinafter. The presentinvention encompasses such multi-epitope vaccines comprisingimmunogenically active peptide fragments of CCL22 optionally incombination with further proteins or peptides fragments not belonging toor derived from CCL22 and/or adjuvants as described hereinafter. Animportant factor driving the development of vaccines having a morecomplex composition is the desire to target multiple tumor antigens e.g.by designing vaccines comprising or encoding a collection of carefullyselected CTL and T_(h) cell epitopes. The invention thus in one aspectrelates to vaccine compositions comprising both Class I and ClassII-restricted CCL22 epitopes.

The peptides of the present invention thus comprise both peptides in ashort ‘MHC-ready’ form (class I restricted), and in a longer formrequiring processing by the proteasome (class II restricted). Thus, thecomposition according to the present invention may be provided as amulti-epitope vaccine comprising class I restricted epitope and/or classII restricted epitopes as defined hereinbefore.

Nucleic Acid Based Vaccine Composition

The vaccine composition according to the present invention may comprisea nucleic acid encoding a CCL22 polypeptide or an immunologically activepeptide fragment thereof. Said nucleic acid may thus encode any of theabove-mentioned proteins and peptide fragments. The nucleic acid may forexample be DNA, RNA, LNA, HNA, PNA, preferably the nucleic acid is DNAor RNA.

The nucleic acids of the invention may be comprised within any suitablevector, such as an expression vector. Numerous vectors are available andthe skilled person will be able to select a useful vector for thespecific purpose. The vector may, for example, be in the form of aplasmid, cosmid, viral particle or artificial chromosome. Theappropriate nucleic acid sequence may be inserted into the vector by avariety of procedures, for example, DNA may be inserted into anappropriate restriction endonuclease site(s) using techniques well knownin the art. Apart from the nucleic acid sequence according to theinvention, the vector may furthermore comprise one or more of a signalsequence, an origin of replication, one or more marker genes, anenhancer element, a promoter, and a transcription termination sequence.The vector may also comprise additional sequences, such as enhancers,poly-A tails, linkers, polylinkers, operative linkers, multiple cloningsites (MCS), STOP codons, internal ribosomal entry sites (IRES) and hosthomologous sequences for integration or other defined elements. Methodsfor engineering nucleic acid constructs are well known in the art (see,e.g., Molecular Cloning: A Laboratory Manual, Sambrook et al., eds.,Cold Spring Harbor Laboratory, 2nd Edition, Cold Spring Harbor, N.Y.,1989). The vector is preferably an expression vector, comprising thenucleic acid operably linked to a regulatory nucleic acid sequencedirecting expression thereof in a suitable cell. Within the scope of thepresent invention said regulatory nucleic acid sequence should ingeneral be capable of directing expression in a mammalian cell,preferably a human cell, more preferably in an antigen presenting cell.

In one preferred embodiment the vector is a viral vector. The vector mayalso be a bacterial vector, such as an attenuated bacterial vector.Attenuated bacterial vectors may be used in order to induce lastingmucosal immune responses at the sites of infection and persistence.Different recombinant bacteria may be used as vectors, for example thebacterial vector may be selected from the group consisting ofSalmonella, Lactococcus], and Listeria. In general, induction ofimmunity to the heterologous antigen HPV16 L1 or E7 could be shown, withstrong CTL induction and tumor regression in mice. The vector mayfurthermore comprise a nucleic acid encoding a T-cell stimulatorypolypeptide.

Loaded APCs

In useful embodiments an immunogenic response directed against a cancerdisease is elicited by administering the peptide of the invention eitherby loading MHC class I or class II molecules on antigen presenting cells(APCs) from the individual, by isolating PBLs from the individual andincubating the cells with the peptide prior to injecting the cells backinto the individual or by isolating precursor APCs from the individualand differentiating the cells into professional APCs using cytokines andantigen before injecting the cells back into the individual.

It is thus an aspect of the invention to provide vaccine compositionscomprising antigen presenting cells comprising CCL22 or animmunologically active peptide fragment thereof or a nucleic acidencoding said protein or said immunologically active peptide fragment.The antigen presenting cell may be any cell capable of presenting anantigen to a T-cell. Preferred antigen presenting cells are dendriticcells. The dendritic cells (DC) may be prepared and used in therapeuticprocedure according to any suitable protocol, for example as describedherein below. It will be appreciated by the person skilled in the artthat the protocol may be adopted to use with individuals with differentHLA type and different diseases.

Dendritic cells (DC) may be pulsed with 50 μg/ml HLA-restricted peptide(synthesized at GMP quality) for 1 h at 37° C. peptide and 5×10⁶ cellsare administered subcutaneously at day 1 and 14, subsequently every 4weeks, additional leukapheresis after 5 vaccinations. The generation ofDC for clinical use and quality control can be performed essentially asdescribed in Nicolette et al., (2007).

Thus, in one embodiment of the present invention, a method for treatingan individual suffering from a clinical condition characterized by theexpression of CCL22, preferably wherein the clinical condition is canceror an infection, is one wherein the peptide is administered bypresenting the peptide to the individual's antigen presenting cells(APCs) ex vivo followed by injecting the thus treated APCs back into theindividual. There are at least two alternative ways of performing this.One alternative is to isolate APCs from the individual and incubate(load) the MHC class I molecules with the peptide. Loading the MHC classI molecules means incubating the APCs with the peptide so that the APCswith MHC class I molecules specific for the peptide will bind thepeptide and therefore be able to present it to T cells. Subsequently,the APCs are re-injected into the individual. Another alternative wayrelies on the recent discoveries made in the field of dendritic cellbiology. In this case, monocytes (being dendritic cell precursors) areisolated from the individual and differentiated in vitro intoprofessional APC (or dendritic cells) by use of cytokines and antigen.Subsequently, the in vitro generated DCs are pulsed with the peptide andinjected into the individual.

Adoptive Immunotherapy/Adoptive Transfer

An important aspect the invention relates to cultivating CCL22 specificT-cells in vitro and adoptive transfer of these to individuals. Adoptivetransfer means that the physician directly transfers the actualcomponents of the immune system that are already capable of producing aspecific immune response, into an individual.

It is one objective to the present invention to provide CCL22 specificT-cells, which may be useful for example for adoptive transfer. IsolatedT-cells comprising T-cell receptors capable of binding specifically toCCL22 peptide/MHC class I or CCL22 peptide/MHC class II complexes can beadoptively transferred to individuals, said T-cells preferably beingT-cells that have been expanded in vitro, wherein the CCL22 peptide maybe any of the immunigenically active peptide fragments of CCL22mentioned herein above. Methods of expanding T-cells in vitro are wellknown to the skilled person. The invention also relates to methods oftreatment comprising administering T-cells comprising T-cell receptorscapable of binding specifically to a MHC-restricted CCL22 peptidecomplex to an individual, such as a human being suffering from a cancerdisease, wherein the CCL22 derived peptide may be any of the CCL22peptides mentioned herein above. The invention furthermore relates touse of T-cells comprising T-cell receptors capable of bindingspecifically to CCL22 or peptide fragments thereof for the preparationof a medicament for the treatment of a cancer or infection. AutologousT-cell transfer may be performed essentially as described in Walter etal., (1995).

TCR Transfer

In yet another embodiment, such T-cells could be irradiated beforeadoptive transfer to control proliferation in the individual. It ispossible to genetically engineer the specificity of T cells by TCR genetransfer (Engels et al., 2007). This allows the transfer of T cellsbearing CCL22 peptide specificity into individuals. In general, the useof T cells for adoptive immunotherapy is attractive because it allowsthe expansion of T cells in a tumor- or virus-free environment, and theanalysis of T cell function prior to infusion.

The application of TCR gene-modified T cells (such as T-cellstransformed with an expression construct directing expressing of aheterologous TCR) in adoptive transfer has several advantages incomparison to the transfer of T cell lines: (i) the generation ofredirected T cells is generally applicable. (ii) High-affinity or veryhigh-affinity TCRs can be selected or created and used to engineer Tcells. (iii) High-avidity T cells can be generated using codon optimizedor murinized TCRs allowing better surface expression of the stabilizedTCRs. Genetic engineering of T cell specificity by T cell receptor (TCR)gene transfer may be performed essentially as described in Morgan et al.(2006).

TCR Transfection

TCR with known anti-tumor reactivity can be genetically introduced intoprimary human T lymphocytes. Genes encoding TCR alpha and beta chainsfrom a tumor specific CTL clone can be transfected into primary T cellsand in this way reprogram T cells with specificity against the tumorantigen. TCR RNA is transfected into PBL by electroporation (Schaft etal., 2006). Alternatively, T cells can be provided with at newspecificity by TCR gene transfer using retroviral vectors (Morgan etal., 2006). However, the provirus from the retroviral vector mightintegrate at random in the genome of the transfected cells andsubsequently disturb cell growth. Electroporation of T cells withTCR-coding RNA overcome this disadvantage, since RNA is only transientlypresent in the transfected cells and cannot be integrated in the genome(Schaft et al., 2006). Furthermore, transfection of cells is routinelyused in the laboratory.

Adjuvants and Carriers

The vaccine composition according to the invention preferably comprisesan adjuvant and/or a carrier. Examples of useful adjuvants and carriersare given herein below. Thus the CCL22 polypeptide, the immunogenicallyactive peptide fragments of CCL22 or functional homologues thereof, thepolypeptides comprising same or nucleic acid encoding same may in acomposition of the present invention be associated with an adjuvantand/or a carrier.

Adjuvants are any substance whose admixture into the vaccine compositionincreases or otherwise modifies the immune response to the CCL22 or toimmunogenically active peptide fragments of CCL22, see further in thebelow. Carriers are scaffold structures, for example a polypeptide or apolysaccharide, to which the CCL22 or peptide fragment thereof iscapable of being associated and which aids in the presentation ofespecially the peptides of the present invention.

Many of the peptides of the invention are relatively small molecules andit may therefore be required in compositions as described herein tocombine the peptides with various materials such as adjuvants and/orcarriers, to produce vaccines, immunogenic compositions, etc. Adjuvants,broadly defined, are substances which promote immune responses. Ageneral discussion of adjuvants is provided in Goding, MonoclonalAntibodies: Principles & Practice (2nd edition, 1986) at pages 61-63.Goding notes, that when the antigen of interest is of low molecularweight, or is poorly immunogenic, coupling to an immunogenic carrier isrecommended. Examples of such carrier molecules include keyhole limpethaemocyanin, bovine serum albumin, ovalbumin and fowl immunoglobulin.Various saponin extracts have also been suggested to be useful asadjuvants in immunogenic compositions. It has been proposed to usegranulocyte-macrophage colony stimulating factor (GM-CSF), a well knowncytokine, as an adjuvant (WO 97/28816).

A carrier may be present independently of an adjuvant. The function of acarrier can for example be to increase the molecular weight of inparticular peptide fragments in order to increase their activity orimmunogenicity, to confer stability, to increase the biologicalactivity, or to increase serum half-life. Furthermore, a carrier may aidin presenting the CCL22 protein, polypeptide, functional homologue orpeptide fragments thereof to T-cells. The carrier may be any suitablecarrier known to a person skilled in the art, for example a protein oran antigen presenting cell. A carrier protein could be, but is notlimited to, keyhole limpet hemocyanin, serum proteins such astransferrin, bovine serum albumin, human serum albumin, thyroglobulin orovalbumin, immunoglobulins, or hormones, such as insulin or palmiticacid. For immunization of humans, the carrier must be a physiologicallyacceptable carrier acceptable to humans and safe. However, tetanustoxoid and/or diphtheria toxoid are suitable carriers in one embodimentof the invention. Alternatively, the carrier may be dextrans for examplesepharose.

Thus it is an aspect of the present invention that the CCL22 protein,polypeptide fragment, variant or peptide derived here from present inthe composition is associated with a carrier such as e.g. a protein ofthe above or an antigen-presenting cell such as e.g. a dendritic cell(DC).

Adjuvants could for example be selected from the group consisting of:AlK(SO₄)₂, AlNa(SO₄)₂, AlNH₄ (SO₄), silica, alum, Al(OH)₃, Ca₃ (PO₄)₂,kaolin, carbon, aluminum hydroxide, muramyl dipeptides,N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-DMP),N-acetyl-nornuramyl-L-alanyl-D-isoglutamine (CGP 11687, also referred toas nor-MDP),N-acetylmuramyul-L-alanyl-D-isoglutaminyl-L-alanine-2-(1′2′-dipalmitoyl-sn-glycero-3-hydroxphosphoryloxy)-ethylamine(CGP 19835A, also referred to as MTP-PE), R1131 (MPL+TDM+CWS) in a 2%squalene/Tween-80® emulsion, lipopolysaccharides and its variousderivatives, including lipid A, Freund's Complete Adjuvant (FCA),Freund's Incomplete Adjuvants, Merck Adjuvant 65, polynucleotides (forexample, poly IC and poly AU acids), wax D from Mycobacterium,tuberculosis, substances found in Corynebacterium parvum, Bordetellapertussis, and members of the genus Brucella, Titermax, ISCOMS, Quil A,ALUN (see U.S. Pat. Nos. 58,767 and 5,554,372), Lipid A derivatives,choleratoxin derivatives, HSP derivatives, LPS derivatives, syntheticpeptide matrixes or GMDP, Interleukin 1, Interleukin 2, Montanide ISA-51and QS-21. Preferred adjuvants to be used with the invention includeoil/surfactant based adjuvants such as Montanide adjuvants (availablefrom Seppic, Belgium), preferably Montanide ISA-51. Other preferredadjuvants are bacterial DNA based adjuvants, such as adjuvants includingCpG oligonucleotide sequences. Yet other preferred adjuvants are viraldsRNA based adjuvants, such as poly I:C. Imidazochinilines are yetanother example of preferred adjuvants. The most preferred adjuvants areadjuvants suitable for human use.

Montanide adjuvants (all available from Seppic, Belgium), may beselected from the group consisting of Montanide ISA-51, MontanideISA-50, Montanide ISA-70, Montanide ISA-206, Montanide ISA-25, MontanideISA-720, Montanide ISA-708, Montanide ISA-763A, Montanide ISA-207,Montanide ISA-264, Montanide ISA-27, Montanide ISA-35, Montanide ISA51F, Montanide ISA 016D and Montanide IMS, preferably from the groupconsisting of Montanide ISA-51, Montanide IMS and Montanide ISA-720,more preferably from the group consisting of Montanide ISA-51. MontanideISA-51 (Seppic, Inc.) is oil/surfactant based adjuvants in whichdifferent surfactants are combined with a non-metabolizable mineral oil,a metabolizable oil, or a mixture of the two. They are prepared for useas an emulsion with an aqueous solution comprising CCL22 or a peptidefragment thereof. The surfactant is mannide oleate. QS-21 (Antigenics;Aquila Biopharmaceuticals, Framingham, Mass.) is a highly purified,water-soluble saponin that handles as an aqueous solution. QS-21 andMontanide ISA-51 adjuvants can be provided in sterile, single-use vials.

The well-known cytokine GM-CSF is another preferred adjuvant of thepresent invention. GM-CSF has been used as an adjuvant for a decade andmay preferably be GM-CSF as described in WO 97/28816.

Desirable functionalities of adjuvants capable of being used inaccordance with the present invention are listed in the below table.

TABLE 3 Modes of adjuvant action Action Adjuvant type Benefit 1. Immuno-Generally small molecules or Upregulation of immune modulation proteinswhich modify the response. Selection of cytokine network Th1 or Th2 2.Presen- Generally amphipathic Increased neutralizing tation molecules orcomplexes which antibody response. interact with immunogen in Greaterduration of its native conformation response 3. CTL Particles which canbind or Cytosolic processing of induction enclose immunogen and proteinyielding correct which can fuse with or disrupt class 1 restrictedpeptides cell membranes w/o emulsions for direct Simple process ifattachment of peptide to cell promiscuous peptide(s) surface MHC-1 known4. Targeting Particulate adjuvants which Efficient use of adjuvant bindimmunogen. Adjuvants and immunogen which saturate Kupffer cellsCarbohydrate adjuvants which As above. May also de- target lectinreceptors on termine type of response macrophages and DCs if targetingselective 5. Depot w/o emulsion for short Efficiency PotentialGeneration term for single-dose Microspheres or nanospheres for vaccinelong term Source: Cox, J. C., and Coulter, A. R. (1997). Vaccine 15,248-56.

A vaccine composition according to the present invention may comprisemore than one adjuvant. Furthermore, the invention encompasses atherapeutic composition further comprising any adjuvant substance and/orcarrier including any of the above or combinations thereof. It is alsocontemplated that the CCL22 protein, variants or peptide fragmentsthereof, and the adjuvant can be administered separately in anyappropriate sequence. Preferably, the vaccine compositions of thepresent invention comprise a Montanide adjuvant such as Montanide ISA 51or Montanide ISA 720 or the GM-CSF adjuvant.

Accordingly, the invention encompasses a therapeutic composition furthercomprising an adjuvant substance including any of the above orcombinations thereof. It is also contemplated that the antigen, i.e. thepeptide of the invention and the adjuvant can be administeredsimultaneously or separately in any appropriate sequence.

Dosis and Administration

The amount of CCL22 or the immunogenically active peptide fragments ofCCL22 of the invention in the vaccine composition may vary, depending onthe particular application. However, a single dose of the peptidecomposition is preferably anywhere from about 10 μg to about 5000 μg,more preferably from about 50 μg to about 2500 μg such as about 100 μgto about 1000 μg. In particular, in embodiments of the invention wherethe individual to be treated is a human being, then a single dose may bein the range of 50 μg to 500 μg, for example in the range of 80 μg to300 μg, such as in the range of 100 μg to 250 μg of CCL22 or saidimmunogenically active peptide fragment of CCL22. Frequently, thevaccine compositions are administered repeatedly over time. For examplethe vaccine composition may be administered at least 2 times, preferablyat least 5 times, more preferably at least 10 times, such as in therange of 10 to 20 times. The vaccine composition may also beadministered continuously. Administration may be repeated at any usefulfrequency. Thus, for example the vaccine compositions may beadministered once every week, such as once every two weeks, for exampleonce every 3 weeks, such as once per month, for example once per twomonths, such as once per three months, for example once per half year,such as once per year. In particular, the vaccine compositions may beadministered continuously. The frequency of administration may alterduring said time. In one embodiment the vaccine compositions areadministered continuously once per 1 to 3 months. Modes ofadministration include intradermal, subcutaneous and intravenousadministration, implantation in the form of a time release formulation,etc. Any and all forms of administration known to the art areencompassed herein. Also any and all conventional dosage forms that areknown in the art to be appropriate for formulating injectableimmunogenic peptide composition are encompassed, such as lyophilizedforms and solutions, suspensions or emulsion forms containing, ifrequired, conventional pharmaceutically acceptable carriers, diluents,preservatives, adjuvants, buffer components, etc.

The pharmaceutical compositions may be prepared and administered usingany conventional protocol known by a person skilled in the art. Inexamples 3-5 non-limiting examples of preparation of a vaccinecomposition according to the invention is given as well as anon-limiting example of administration of such as a vaccine. It will beappreciated by the person skilled in the art that the protocol may beeasily adapted to any of the vaccine compositions described herein. In afurther embodiment of the invention, the pharmaceutical composition ofthe invention is useful for treating an individual suffering from aclinical condition characterized by expression of CCL22, such as cancerand infections.

The immunoprotective effect of the composition of the invention can bedetermined using several approaches known to those skilled in the art. Asuccessful immune response may also be determined by the occurrence ofDTH reactions after immunization and/or the detection of antibodiesspecifically recognizing the peptide(s) of the vaccine composition.

Vaccine compositions according to the invention may be administered toan individual in therapeutically effective amounts. The effective amountmay vary according to a variety of factors such as the individual'scondition, weight, sex and age. Other factors include the mode ofadministration.

The pharmaceutical compositions may be provided to the individual by avariety of routes such as subcutaneous, topical, oral and intramuscular.Administration of pharmaceutical compositions is accomplished orally orparenterally. Methods of parenteral delivery include topical,intra-arterial (directly to the tissue), intramuscular, subcutaneous,intramedullary, intrathecal, intraventricular, intravenous,intraperitoneal, or intranasal administration. The present inventionalso has the objective of providing suitable topical, oral, systemic andparenteral pharmaceutical formulations for use in the methods ofprophylaxis and treatment with the vaccine composition.

For example, the vaccine compositions can be administered in such oraldosage forms as tablets, capsules (each including timed release andsustained release formulations), pills, powders, granules, elixirs,tinctures, solutions, suspensions, syrups and emulsions, or byinjection. Likewise, they may also be administered in intravenous (bothbolus and infusion), intraperitoneal, subcutaneous, topical with orwithout occlusion, or intramuscular form, all using forms well known tothose of ordinary skill in the pharmaceutical arts. An effective butnon-toxic amount of the vaccine, comprising any of the herein describedcompounds can be employed as a prophylactic or therapeutic agent. Alsoany and all conventional dosage forms that are known in the art to beappropriate for formulating injectable immunogenic peptide compositionare encompassed, such as lyophilized forms and solutions, suspensions oremulsion forms containing, if required, conventional pharmaceuticallyacceptable carriers, diluents, preservatives, adjuvants, buffercomponents, etc.

Preferred modes of administration of the vaccine composition accordingto the invention include, but are not limited to systemicadministration, such as intravenous or subcutaneous administration,intradermal administration, intramuscular administration, intranasaladministration, oral administration, rectal administration, vaginaladministration, pulmonary administration and generally any form ofmucosal administration. Furthermore, it is within the scope of thepresent invention that the means for any of the administration formsmentioned in the herein are included in the present invention.

A vaccine according to the present invention can be administered once,or any number of times such as two, three, four or five times.Administering the vaccine more than once has the effect of boosting theresulting immune response. The vaccine can further be boosted byadministering the vaccine in a form or body part different from theprevious administration. The booster shot is either a homologous or aheterologous booster shot. A homologous booster shot is a where thefirst and subsequent vaccinations comprise the same constructs and morespecifically the same delivery vehicle especially the same viral vector.A heterologous booster shot is where identical constructs are comprisedwithin different viral vectors.

Second Active Ingredient

It is an aspect of the present invention that the vaccine compositionherein provided is used in combination with a second active ingredient.The administration of the vaccine composition and the second activeingredient may be sequential or combined. Examples of second activeingredients are given above for both cancers and infections. It is afurther aspect that the vaccine composition may be used in combinationwith other therapy of relevance for the given clinical condition to betreated. Such therapy may include surgery, chemotherapy or gene therapy,immunostimulating substances or antibodies; a person skilled in the artis able to determine the appropriate combination treatment for a givenscenario.

In some cases it will be appropriate to combine the treatment method ofthe invention with a further medical treatment such as chemotherapy,radiotherapy, treatment with immunostimulating substances, gene therapy,treatment with antibodies and/or antibiotics and treatment usingdendritic cells.

Monitoring Immunization

In preferred embodiments, the pharmaceutical composition of theinvention is a vaccine composition. It is therefore of interest, and anaspect of the present invention to monitor the immunization in anindividual to whom the vaccine composition of the present invention isadministered. The pharmaceutical composition may thus be an immunogeniccomposition or vaccine capable of eliciting an immune response to acancer and/or infection. As used herein, the expression “immunogeniccomposition or vaccine” refers to a composition eliciting at least onetype of immune response directed against CCL22 expressing cells such ascancer cells, APCs or DCs. Thus, such an immune response may be any ofthe following: A CTL response where CTLs are generated that are capableof recognizing the HLA/peptide complex presented on cell surfacesresulting in cell lysis, i.e. the vaccine elicits the production in thevaccinated subject of effector T-cells having a cytotoxic effect againstthe cancer cells; a B-cell response giving rise to the production ofanti-cancer antibodies; and/or a DTH type of immune response. It is onobject of the present invention to monitor the immunization of anindividual by monitoring any of the above reactions subsequent toadministering the composition of the present invention to saidindividual.

In one aspect the invention relates to methods of monitoringimmunization, said method comprising the steps of

i) providing a blood sample from an individual

ii) providing CCL22 or a peptide fragment hereof, wherein said proteinor peptide may be any of the proteins or peptides described herein

iii) determining whether said blood sample comprises antibodies orT-cells comprising T-cell receptors specifically binding the protein orpeptide

iv) thereby determining whether an immune response to said protein orpeptide has been raised in said individual.

The individual is preferably a human being, for example a human beingthat has been immunized with CCL22 or immunogenically active peptidefragments of CCL22 or a nucleic acid encoding said protein or peptide.

Kit of Parts

The invention also relates to a kit-of-parts comprising

-   -   any of the vaccine compositions described herein and/or    -   a CCL22 protein or functional homologue hereof and/or    -   any of the immunogenically active peptide fragments of CCL22,        functional homologues hereof, and/or peptides derived here from        as described herein and/or    -   any of the nucleic acids encoding the proteins of the above two        bullet points        and instructions on how to use the kit of parts.

The invention also relates to a kit-of-parts comprising

-   -   any of the vaccine compositions described herein and/or    -   a CCL22 protein or functional homologue hereof and/or    -   any of the immunogenically active peptide fragments of CCL22,        functional homologues hereof, and/or peptides derived here from        as described herein and/or    -   any of the nucleic acids encoding the proteins of the above two        bullet points        and a second active ingredient.

Preferably, the second active ingredient is chosen in correspondencewith the clinical condition to be treated so that in the case where acancer is to be treated the second active ingredient is chosen amonge.g. chemotherapeutic agents as listed above. Likewise, if treating amicrobial/viral infection, the second active ingredient is preferably ananti-biotic and/or an anti-viral agent.

The components of the kit-of-parts are preferably comprised inindividual compositions, it is however within the scope of the presentinvention that the components of the kit-of-parts all are comprisedwithin the same composition. The components of the kit-of-parts may thusbe administered simultaneously or sequentially in any order.

SEQUENCE LISTING

SEQ ID NO Description SEQ ID NO: 1 Polypeptide fragment of human CCL22,CCL22₁₋₂₂ SEQ ID NO: 2 Polypeptide fragment of human CCL22, CCL22₇₋₁₅SEQ ID NO: 3 Polypeptide fragment of human CCL22, CCL22₃₋₁₁ SEQ ID NO: 4Polypeptide fragment of human CCL22, CCL22₃₋₁₂ SEQ ID NO: 5 Polypeptidefragment of human CCL22, CCL22₁₁₋₁₉ SEQ ID NO: 6 Polypeptide fragment ofhuman CCL22, CCL22₅₋₁₃ SEQ ID NO: 7 Polypeptide fragment of human CCL22,CCL22₁₄₋₂₂ SEQ ID NO: 8 Polypeptide fragment of human CCL22, CCL22₈₋₁₇SEQ ID NO: 9 Polypeptide fragment of human CCL22, CCL22₉₋₁₇ SEQ ID NO:10 Polypeptide fragment of human CCL22, CCL22₆₋₁₅ SEQ ID NO: 11Polypeptide fragment of human CCL22, CCL22₁₋₂₄ SEQ ID NO: 12 Amino acidsequence of CCL22 of Homo sapiens SEQ ID NO: 13 mCL22long SEQ ID NO: 14mCL22short SEQ ID NO: 15 Full length amino acid sequence of murineCCL22, Uniprot accession number O88430 SEQ ID NO: 16 Consensus sequence

EXAMPLES

The invention is further illustrated by the following examples, whichshould however not be construed as limiting for the invention.

Example 1—Methods

PBMC—Patient Material

Peripheral Blood Mononuclear Cells (PBMC) were collected from healthyindividuals and cancer patients (melanoma, renal cell carcinoma andbreast cancer patients). Blood samples were drawn a minimum of fourweeks after termination of any kind of anti-cancer therapy. PBMC wereisolated using Lymphoprep™ separation, HLA-typed and frozen in FCS with10% DMSO. Fresh ovarian ascites was filtered with a 70 μm filter and thecells were isolated by centrifugation (1500 RPMI-1640, 5 min). If theascites contained a high abundance of erythrocytes, they were removed byadding lysis-buffer (Ortho-Mune Lysing Solution) to the cells withincubation of 3-5 min. The lysis-buffer was quickly washed away and thecells cryopreserved in human serum with 10% DMSO at −140 degrees untiluse. The protocol was approved by the Scientific Ethics Committee forThe Capital Region of Denmark and conducted in accordance with theprovisions of the Declaration of Helsinki. Written informed consent fromthe patients was obtained before study entry.

Establishment of Antigen-Specific T-Cell Cultures

CCL22-specific T cell cultures were established by stimulation of cancerpatient PBMC with irradiated pCCL22₃₋₁₂ peptide-loaded autologous DC orPBMCs. The following day IL-7 and IL-12 (PeproTech, London, UK) wereadded. Stimulation of the cultures were carried out every 8 days withCCL22₃₋₁₂ peptide loaded irradiated autologous DC followed by pCCL22₃₋₁₂peptide-loaded irradiated autologous PBMC. The day after peptidestimulation IL-2 (PeproTech, London, UK) was added. 4 DC stimulationsand 1 PBMC stimulation were made in total.

Generation of DC

DC were generated from PBMC by adherence on culture dishes at 37° C. for1-2 hr. in RPMI-1640. Adherent monocytes were cultured in RPMI-1640supplemented with 10% fetal calf serum in the presence of IL-4 (250U/ml) and GM-CSF (1000 U/ml) for 6 days. DC were matured by addition ofIL-13 (1000 U/ml), IL-6 (1000 U/ml) TNF-α (1000 U/ml) and PGE₂ (1ug/ml).

Cytotoxicity Assay

Conventional ⁵¹Cr-release assays for CTL-mediated cytotoxicity werecarried out as described elsewhere (Andersen et al., 1999). Target cellswere T2-cells (ATCC), HLA-A2⁺ EBV transformed B-cell line (RPM16666),AML cells (UKE-1 and THP1) colon cancer cells (SW480), melanoma cellline (FM55-M2), Breast cancer cells MDA-MB231 with or without IFN-γ (100U/ml) addition for 2 days prior to performing cytotoxicity assay.

For peptide titration cytotoxicity assay, T2 cells were used as targetcells and a constant effector to target ratio of 3:1 was used for allpeptide concentrations. 10-fold serial peptide dilutions ranging from10-2 mM to 10-9 mM of pCCL22₃₋₁₂ and pCCL22₃₋₁₁ were made.

siRNA Mediated CCL22 Silencing

A set of three Stealth siRNA duplexes for targeted silencing of CCL22(HSS109578, HSS184551, HSS184552) were obtained from Invitrogen(Invitrogen, Paisley, UK). For CCL22 silencing experiments, THP-1 cellswere transfected with CCL22 siRNA using electroporation parameters aspreviously described (Met et al., 2011; Hobo et al., 2010).

Flow Cytometric Analysis

Flow cytometry analysis was performed on a FACSCanto™ II (BDBiosciences, San Jose Calif., USA), cell sorting was performed onFACSAria™ (BD Biosciences, San Jose Calif., USA).

Intracellular staining of CCL22-specific T cell cultures was performedafter the cells were stimulated with HIV or pCCL22₃₋₁₂ peptides for 5hours (BD GolgiPlug™ was added after the first hour). The cells werethen stained for surface markers, then washed and permeabilized by usingFixation/Permeabilization and Permeabilization Buffer (eBioscience),according to manufacturer's instructions. Antibodies used: IFNγ-PE-Cy7,TNFα-APC, CD4-PerCP, CD8-FITC (all from BD Biosciences). Dead cells werestained using LIVE/DEAD® Fixable Near-IR Dead Cell Stain Kit accordingto manufacturer's instructions.

For tetramer staining and sorting PE and APC coupled HLA-A2 multimerswith HIV or pCCL22₃₋₁₂ peptides were used in addition to the CD4-PerCPand CD8-FITC (BD Biosciences). HLA-A2 multimers were produced in houseby using a previously described MHC peptide exchange technology (15).

ELISPOT Assay

In the present study the ELISPOT was performed according to theguidelines provided by CIP(http://cimt.eu/cimt/files/dl/cip_guidelines.pdf). The ELISPOT assay wasused to quantify peptide epitope specific effector cells that releasecytokines (IFNγ, TNFα, IL-17A or IL-10) as described previously(Sorensen et al., 2012). In some experiments, PBMCs were stimulated oncein vitro with peptide prior to analysis. In some experiments, 10⁴autologous DCs were added to the wells as antigen presenting cells. Thespots were counted using the ImmunoSpot Series 2.0 Analyzer(C.T.L.-Europe, Bonn, Germany). In some experiments, CD4⁺ cells wereisolated by EasySep human CD4⁺ T cell enrichment kit (Stem Celltechnologies, Grenoble, France) following manufacturers' instructions.This yielded highly pure cultures (>97% CD4+), which was confirmed bystaining with surface antibodies as described below for intracellularcytokine staining (ICS) and flow cytometry (FCM).

PBMCs were placed in the bottom of ELISPOT plate (nitrocellulosebottomed 96-well plates by MultiScreen MAIP N45; Millipore) pre-coatedwith IFN-γ capture Ab (Mabtech) and the peptides were added at 5 μg/ml.PBMCs from each patient were set up in duplicates or triplicates forpeptide and control stimulations. Cells were incubated in ELISPOT platesin the presence of an antigen for 14-16 hours after which they arewashed off and secondary biotinylated Ab (Mabtech) was added. After 2 hincubation unbound secondary antibody was washed off and streptavidinconjugated alkaline phosphatase (AP) (Mabtech) was added for 1 h. Next,unbound conjugated enzyme is washed off and the assay is developed byadding BCIP/NBT substrate (Mabtech). Developed ELISPOT plates wereanalysed on CTL ImmunoSpot S6 Ultimate-V analyzer using Immunospotsoftware v5.1. Responses were calculated as the difference betweenaverage numbers of spots in wells stimulated with pCCL22₃₋₁₂ peptide andcontrol wells.

Analysis of CCL22 Expression

Cell culture supernatants from PBMC, cancer cell lines and ascites cellcultures as well as siRNA transfected THP-1 cells were analyzed usingHuman CCL22/MDC DuoSet ELISA kit (R&D Systems) according tomanufacturer's instructions.

Peptide Stimulation of PBMCs and Ascites Cells

PBMCs from healthy donors or cancer patients were thawed and rested for4 hours in X-VIVO 15™ (Lonza) before being set up into 24-well plateswith 2×10⁶ cells/well in X-Vivo medium with 5% human serum. 20 μg/ml ofpCCL22₃₋₁₂ peptide in DMSO or HIV peptide in sterile water were added toeach well. Appropriate amount of DMSO was added to HIV control wells tocontrol for the solvent of pCCL22₃₋₁₂ peptide. The following day, IL-2was added to a final concentration of 120 U/ml (360 U for ascites cellsstimulation). Supernatant samples were collected after 2 or 7 days ofculture. Cells were stimulated twice before being tested in ELISPOTassay.

Cytokine Expression LUMINEX

Cell culture supernatants from PBMCs or ascites cells stimulated withCCL22-3 or HIV peptide were analyzed for IFN-γ, TNF-α, IL-6, IL-10 andIL-1β using Bio-Plex Pro™ Human Chemokine assays from Bio-Rad. Sampleswere acquired on Bio Plex 200 system and analyzed using Bio-PlexManager™ v6.

Statistical Analysis

t-test was used for the statistical analysis of ELISA samples fromcancer patients and healthy donors when comparing pCCL22₃₋₁₂ stimulationwith HIV control. The same analysis was used to compare IFN-γ, TNF-α,IL-6, IL-10 expression after pCCL22₃₋₁₂ stimulation with HIV control.Wilcoxon-signed rank test was used for statistical analysis of IL-10expression due to multiple zero values.

Example 2—Results

A panel of CCL22-derived HLA-binding peptides was predicted usingSYFPEITHI database (www.syfpeithi.de):

CCL22-1 ALLVVLVLL (7-15) CCL22-2 RLQTALLVV (3-11) CCL22-3 RLQTALLVVL(3-12) CCL22-4 VLVLLAVAL (11-19) CCL22-5 QTALLVVLV (5-13) CCL22-6LLAVALQAT (14-22) CCL22-7 LLVVLVLLAV (8-17) CCL22-8 LVVLVLLAV (9-17)CCL22-9 TALLVVLVL (6-15) CCL22 LONG MDRLQTALLVVLVLLAVALQAT (1-22)CCL22-SIGNAL MDRLQTALLVVLVLLAVALQATEA (1-24)

PBMCs from cancer patients were screened for responses against thesepeptide epitopes by IFNγ ELISPOT. Indications for specific responsesagainst CCL22 peptide CCL22-3 (SEQ ID NO: 4) were found in a number ofmelanoma patients.

PBMCs from a melanoma patient were stimulated with autologous DCs pulsedwith the CCL22-3 peptide. After 5 stimulations the T cell culture wasfound to specifically recognize and lyse CCL22-3 pulsed T2 cells (FIG.3A).

CCL22 specific cells were then isolated by tetramer staining andexpanded, obtaining a clear population of CCL22-3:tetramer positivepopulation (FIG. 2B).

The nature and reactivity of CCL22 specific T cells was evaluated byintracellular staining and it was found that CD8+ T cells were secretingIFN-γ and TNF-α in response to CCL22-3 peptide stimulation, whereas noreactivity was seen from the CD4+ T cells (FIG. 2A).

HLA-A2-restricted reactivity of CCL22-specific T cells was shown byusing HLA-A2 negative and stably transfected K562 cells as targets in achromium release assay (FIG. 3).

CCL22-specific cultures were tested for reactivity against severaldifferent cancer cells, i.e. leukemia cells (THP-1, RPM16666, UKE-1),colon cancer (SW480) as well as breast cancer (MDA).

T cells reactive to CCL22-3 were able to recognize and lyze CCL22expressing cancer cell lines, as shown by chromium release assay (FIG.4). We also found that CCL22-peptide specific cells were able to lysevarious cancer cell lines with and without treatment with IFN-γ (FIG.4).

The killing of target cells by CCL22-specific T cells was dependent ofthe CCL22 expression by the target cells. Thus, decreased CCL22expression by siRNA transfection of THP-1 cells decreased the T-cellmediated lysis (FIG. 5).

Example 3—CCL22-Specific T Cells

We screened the amino acid sequence of human CCL22 protein for possibleHLA-A2-binding peptide epitopes using the SYFPEITHI epitope predictionalgorithm available at www.syfpeithi.de. Interestingly, all high-scoringepitopes were located in the signal peptide portion of the sequence,which is cleaved off before the protein is secreted. One of thehigh-scoring CCL22-derived peptides was RLQTALLVVL, hereafter referredto as pCCL22₃₋₁₂. To characterize pCCL22₃₋₁₂-specific T cells, weacquired PBMCs from a melanoma patient, and stimulated these cells usingautologous DCs or PBMCs pulsed with the pCCL22₃₋₁₂ peptide. After fivestimulations, we performed pCCL22₃₋₁₂-tetramer staining, which revealedsmall but distinct population of tetramer-positive cells (FIG. 2A,left). The tetramer-positive populations were successfully isolated andexpanded using the rapid expansion protocol (FIG. 2B, right). On theexpanded culture, we performed intracellular staining for INFγ and TNFαin response to peptide stimulation. Around 30% of CD8⁺ cells secretedIFNγ and TNFα in response to pCCL22₃₋₁₂ peptide stimulation (FIG. 2A).Only CD8⁺ T cells secreted INFγ and TNFα in response to the pCCL22₃₋₁₂peptide, with no response detected from CD4+ T cells. The same resultswere found with intracellular staining before tetramer isolation andexpansion (data not shown).

Example 4—pCCL22₃₋₁₂-Specific T Cells Exhibit HLA-A2-Restricted Killing

Next, we examined the cytotoxic capability of CCL22-specific T cells.The pCCL22₃₋₁₂-specific T cells were able to lyse pCCL22₃₋₁₂-pulsed T2cells, but did not recognize T2 cells pulsed with a negative controlpeptide from HIV (FIG. 3A). To confirm the HLA-A2-restricted reactivityof CCL22-specific T cells, we used HLA-A2-transfected K562 cells pulsedwith and without pCCL22₃₋₁₂ as targets (FIG. 3B), and found that the Tcells only recognized the K562-A2 cells pulsed with pCCL22₃₋₁₂. As anadditional control, we examined non-transfected HLA-negative K562 cells,determining that these cells were not recognized by thepCCL22₃₋₁₂-specific T cells even when pulsed with pCCL22₃₋₁₂.

We further examined whether the pCCL22₃₋₁₂ epitope could becross-presented from the 22-mer signal peptide sequence of the CCL22protein (termed the CCL22-signal peptide; MDRLQTALLVVLVLLAVALQAT).Indeed, pCCL22₃₋₁₂-specific T cells lysed the T2 cells that were pulsedwith CCL22-signal peptide (FIG. 3C), indicating that T2 cells couldcross-present the pCCL22₃₋₁₂ epitope even without TAP expression inthese cells. We then examined the T-cell avidity of pCCL22₃₋₁₂-specificT cells towards the pCCL22₃₋₁₁ peptide (RLQTALLVV), which is one aminoacid shorter than pCCL22₃₋₁₂ and was predicted by the computer algorithmto bind to HLA-A2 with high affinity. Although the T cells reactedtowards both peptides, they showed the highest avidity towards thedecamer pCCL22₃₋₁₂ epitope (FIG. 3D).

Example 5—Cytotoxicity Against CCL22-Expressing Cancer Cells

The CCL22-specific cytotoxic T-cell lymphocyte (CTL) culture was testedfor reactivity against the leukemia cell lines THP-1, RPM16666, UKE-1,and SET-2; the colon cancer cell line SW480; the breast cancer cell lineMDA; and the melanoma cell line FM55-M2. PCR analysis revealed CCL22expression in all of these cell lines except for FM55-M2 (data notshown), although SET-2 showed only a weak band. We found thatpCCL22₃₋₁₂-specific cells lysed most of the investigated cancer celllines, both with and without pre-treatment with interferon-gamma (IFN-γ)(FIG. 4A-E). IFN-γ reportedly induces CCL22 expression in cancer celllines, and increases surface expression of HLA. Chromium release assayrevealed that SET-2 cells were not killed, which were only weaklypositive for CCL22 mRNA on PCR (FIG. 4F). Additionally, the melanomacell line FM55-M2 showed no CCL22 mRNA on PCR, and these cells were notlysed by CCL22-specific CTLs (data not shown). To confirm that thekilling of cancer cells by CCL22-specific CTLs was indeed dependent onCCL22 expression, we used siRNA transfection to inhibit CCL22 expressionin IFNγ pre-treated THP-1 cells. This transfection rescued these cellsfrom T-cell mediated lysis (FIG. 4G). FIG. 4H depicts the CCL22inhibition in THP-1 cells after siRNA transfection as measured by ELISAfrom the supernatant.

Example 6—Spontaneous T-Cell Responses Against CCL22

We next acquired PBMCs from 13 cancer patients and 10 healthyindividuals, and stimulated these cells with the pCCL22₃₋₁₂ peptide fortwo weeks in the presence of low-dose IL-2. We used the IFN-γEnzyme-Linked ImmunoSPOT (ELISPOT) assay to analyze the reactivitytowards the pCCL22₃₋₁₂ peptide. Spontaneous specific T-cell reactivityagainst pCCL22₃₋₁₂ was detected in a number of melanoma patients andhealthy donors (FIG. 8A). Notably, the overall responses appear to besimilar in healthy donors and cancer patients (FIG. 8B).

Example 7—T-Cell-Mediated Decrease in CCL22 Levels in theMicroenvironment

Donor PBMCs that showed a pCCL22₃₋₁₂ response in ELISPOT were thenstimulated twice with pCCL22₃₋₁₂ peptide in the presence of IL2. Next,the culture was depleted of pCCL22₃₋₁₂-reactive T cells usingHLA-A2/pCCL22₃₋₁₂-tetramer and magnetic beads. This T-cell-depletedculture was divided into two portions, and theHLA-A2/pCCL22₃₋₁₂-tetramer-isolated T cells were added back to one ofthe portions (FIG. 8C). We then monitored the CCL22 concentration in thesupernatants of both cultures. Notably, CCL22 levels were lower in theculture with added pCCL22₃₋₁₂-specific T cells after only one day, andthis difference increased over the culturing time. After nine days ofculture, the CCL22 concentration was almost three times higher in thetetramer-depleted culture compared to in the tetramer-enriched culture(FIG. 8D).

Example 8—pCCL22₃₋₁₂ Stimulation Decreased the CCL22 Levels in theMicroenvironment

To mimic a setting in which cancer patients are vaccinated withCCL22-derived peptides, we stimulated PMBCs with the pCCL22₃₋₁₂ peptideepitope in vitro and IL-2. We then investigated whether this activationof pCCL22₃₋₁₂-specific T cells affected the overall CCL22 concentrationamong the PMBCs. First, donor PBMCs that showed a pCCL22₃₋₁₂ response inELISPOT, were stimulated using the pCCL22₃₋₁₂ peptide, and we measuredthe CCL22 concentration in the supernatant over one week (FIG. 9A).CCL22 expression was lower in the cultures stimulated with pCCL22₃₋₁₂peptide compared to cultures stimulated with an HIV control peptide.This difference was apparent after two days of culture and reachedsignificance after one week (P=0.01).

We subsequently used pCCL22₃₋₁₂ peptide or an HIV control peptide tostimulate PBMCs from 11 healthy donors and 13 cancer patients, and thenmeasured the CCL22 concentration in the supernatants one week afterstimulation. In PBMCs from healthy donors, the CCL22 concentrationsignificantly decreased following stimulation with pCCL22₃₋₁₂ peptide(P=0.02) (FIG. 9B). On the other hand, in PBMCs from cancer patients,the overall decrease in CCL22 concentration after stimulation withpCCL22₃₋₁₂ did not reach significance (P=0.17) (FIG. 9C). When PBMCsfrom cancer patients were stratified according to low CCL22 expression(≤2000 pg/mL) and high CCL22 expression (≥5000 pg/mL) (FIG. 9C), thehigh-expression group showed a significant decrease in CCL22concentration after pCCL22₃₋₁₂ stimulation (P=0.005).

Ovarian ascetic fluid reportedly contains a mixture of cancer cells andimmune-infiltrating cells, along with high levels of CCL22. To examinewhether pCCL22₃₋₁₂-specific T cells may influence CCL22 concentrationdirectly in the tumor microenvironment, we collected ascetic fluid fromfive patients with HLA-A2-positive epithelial ovarian cancer, andisolated the ascites cells. The ascites cells from two of these patientsshowed low viability and, thus, we could only analyze cells from threepatients. The ascites cells from one of these patients did not includeany T-lymphocytes. The ascites cells from the remaining two ovariancancer patients were stimulated with pCCL22₃₋₁₂ peptide, which led to adecrease in the overall CCL22 levels in the supernatants at one weekafter stimulation (FIG. 9D).

Example 8—pCCL22₃₋₁₂ Stimulation Influenced the Cytokine Milieu

We further examined the PBMC supernatants from 11 cancer patients and 10healthy donors with regards to changes in cytokine levels after one weekof stimulation with pCCL22₃₋₁₂ peptide compared to with an HIV controlpeptide. The PBMCs from cancer patients that were stimulated with CCL22peptide showed a significant increase in IL-6 level (P=0.02). A similarincrease was observed in cultures of PBMCs from healthy donors, althoughthis change did not reach significance (P=0.06) (FIG. 10A). We alsoobserved a tendency of decreasing TNFα levels in cultures of PBMCs fromhealthy donors (7 out of 10) and cancer patients (7 out of 11); however,these changes did not reach significance (P=0.7 and P=0.16,respectively) (FIG. 10B). We further examined the concentrations ofIL-1β, IL-10, and IFN-γ in the culture supernatants. We detected nounambiguous differences in these cytokines between cultures stimulatedwith pCCL22₃₋₁₂ peptide versus control peptide. After stimulation, IL-10was almost undetectable in the supernatants and IL-113 was induced afterstimulation with pCCL22₃₋₁₂ in only one cancer patient and two healthydonors (data not shown).

Example 9—Conclusion

Our present findings demonstrated that it was possible for specific Tcells to target CCL22-expressing cells.

We demonstrated that it was possible for T cells to recognize anHLA-restricted CCL22-derived peptide epitope, and we were thus able toexpand CCL22-specific T cells by re-stimulation of PBMCs with a CCL22peptide in vitro. The results of our chromium release cytotoxicityassays further demonstrated specific recognition and lysis ofCCL22-expressing cancer cells (breast and colon cancer cells, andleukemia cells). Moreover, CCL22 knockdown by siRNA transfection rescuedcells from being killed by CCL22-specific T-cells. These findingssuggest that in CCL22-expressing cells, the signal peptide is degradedand the epitope is subsequently processed and presented on the cellsurface restricted to HLA-A2 molecules. We also found that the CCL22signal peptide could be taken up and cross-presented on the surface ofnon-professional antigen-presenting cells.

We used the ELISPOT assay to examine PBMCs from HLA-A2⁺ cancer patientsand healthy individuals for reactivity against the CCL22-derived T-cellepitope, and found that T cells spontaneously reacted to theCCL22-derived peptide. Tetramer enrichment/depletion experimentsrevealed that the addition of HLA-A2-restricted CCL22-specific T cellsto PBMCs decreased the CCL22 level in the microenvironment. We furtherdetermined that activation of CCL22-specific T cells via stimulationwith the peptide epitope significantly decreased CCL22 levels amongPMBCs from both healthy donors and cancer patients with high CCL22production. Such activation also led to a CCL22 decrease in supernatantsof ascites-derived cells isolated from ovarian cancer patients. Thesefindings show that CCL22-specific T cells may be used to targetCCL22-expressing cells, and to thereby suppress CCL22-mediated Tregmigration into the tumor microenvironment.

Interestingly, activating CCL22-specific T cells by peptide stimulationalso resulted in increased release of IL-6 into the PMBC supernatants,showing that CCL22-specific T cells may influence the overallpro-inflammatory microenvironment. ELISPOT results showed spontaneousCCL22-specific T-cell responses in both cancer patients and healthydonors, which was somewhat surprising since CCL22 is abundantlyexpressed in normal immune cells.

In conclusion, our present results show that CD8+ T cells couldrecognize an HLA-restricted CCL22 peptide epitope. These T cellsrecognized and lysed various cancer cell lines in a manner dependent onCCL22 expression, and were naturally present in cancer patients andhealthy individuals. Activation of CCL22-specific T cells may directlyinfluence the CCL22 concentration in the microenvironment.

Example 10—Murine CCI22 Peptide Vaccination of C57BL/6

Mice

Female C57BL/6 mice were obtained from Taconic M&B (Denmark) or Janvier(France). All mice were acclimatized for at least one week prior to theinitiation of each experiment. Mice entered experiments at ˜8-14 weeksof age. The experimental procedures were approved by the national ethicscommittee on experimental animal welfare and performed according to theDanish guidelines.

Peptides

Two different murine CCL22 peptides: one long peptide, here namedmCCL22Long (MATLRVPLLVALVLLAVAIQTS) and one short peptide, here namedmCCL22short (VALVLLAVAI; part of mCCL22Long) were synthesized by TAGCopenhagen (Copenhagen, Denmark).

Peptide Vaccination

Murine CCL22 peptide (TAG Copenhagen, Denmark) stocks of 10 mM(mCCL22Short) and 5 mM (mCCL22Long) were made in DMSO. Mice werevaccinated subcutaneously (s.c) on the lower back with 100 μg peptide in1:1 (vol/vol) IFA/PBS emulsion in a total volume of 100 μl. Mice werevaccinated once a week for 3 weeks and sacrificed one week after thelast vaccination.

Preparation of Splenocyte Single Cell Solution

Spleens from sacrificed mice were removed and smashed through a cellstrainer (0.7 μm) and washed with R10 media: RPMI 1640 (Gibco® by LifeTechnologies™) with 10% FCS (Gibco® by Life Technologies™). Red bloodcells lysed by adding red blood cell lysis buffer for 2 min, followed bytwo washes with R10 with 10% fetal bovine serum.

ELISPOT Assay

ELISPOT was performed as described by the manufacturer's instruction. Inshort, 96-well MSIPN4W ELISPOT plate (Millipore) were coated with mouseIFNy-specific capture Ab (AN18; Mabtech) in a concentration of 12 μg/mlin PBS overnight at 4° C. 8×10⁵ or 4×10⁵ splenocytes/well were seeded inthe plate in triplicates. To investigate IFNy response to murine CCL22peptides, cells were incubated with different peptides (5 μM) or R10 ascontrol for 18-20 h at 37° C. ELISPOT was developed with mouseIFNy-specific detection Ab (R4-6A2-biotin; Mabtech) in a concentrationof 1 μg/ml in Buffer (PBS, 0.5% BSA and NaN₃) for 2 h at roomtemperature, followed by 6 washes in PBS. Next, adding ofstreptavidin-ALP (1:1000; Mabtech) in Buffer for 1 h at roomtemperature. Spots were developed by adding substrate solution BCIP/NBT(Mabtech) and stopped by washing in tap water.

ELISPOT were analyzed by ELISPOT Reader (CTL-Immunospot). The resultsare presented in FIG. 11, and show that an immune response can be raisedin vivo using the peptides of the present disclosure.

REFERENCES

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The invention claimed is:
 1. An immunogenically active peptide fragmentof CCL22 consisting of at the most 50 consecutive residues of SEQ ID NO:12, wherein said peptide fragment is of at least 25 amino acids andcontains a sequence selected from the group consisting of SEQ ID NO: 1,SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO:
 11. 2. Thepeptide fragment according to claim 1, wherein the peptide fragmentcomprises the sequence of SEQ ID NO:
 5. 3. A composition comprising thepeptide fragment of claim 1 and an adjuvant.
 4. The compositionaccording to claim 3, further comprising an immunogenically activeprotein or peptide fragment selected from a protein or peptide fragment,which is not CCL22.
 5. The composition according to claim 3, wherein theadjuvant is selected from the group consisting of bacterial DNA basedadjuvants, oil/surfactant based adjuvants, viral dsRNA based adjuvantsand imidazochinilines.